Restoration of tumor suppression using mrna-based delivery system

ABSTRACT

Compositions and methods for treating cancer that include administering a therapeutically effective amount of a tumor suppressor mRNA complexed with a delivery vehicle as described herein, e.g., a nanoparticle.

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Application Ser.No. 62/419,654, filed on Nov. 9, 2016. The entire contents of theforegoing are incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos.HL127464 and CA200900 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

TECHNICAL FIELD

Described herein are compositions and methods for treating cancer thatinclude administering a tumor suppressor mRNA complexed with a deliveryvehicle as described herein, e.g., a nanoparticle.

BACKGROUND

The loss or mutation of tumor suppressors contributes to the developmentand progression of tumors. Stable restoration of tumor suppression hasbeen difficult to achieve.

SUMMARY

Loss and/or mutation of tumor-suppressor genes is a dominant force intumor development and clinical resistance to a variety of therapies;see, e.g., McGillicuddy et al., Cancer Cell. 2009 Jul. 7; 16(1):44-54;Radtke and Raj, Nat Rev Cancer. 2003 October; 3(10):756-67; Goodrich,Oncogene. 2006 Aug. 28; 25(38):5233-43; Seton-Rogers, Nat Rev Cancer.2009 September; 9(9):610; Juric et al., Nature. 2015 Feb. 12;518(7538):240-4; and Peng et al., Cancer Discov. 2016 February;6(2):202-16. A wide range of tumor suppressors (Table 1) has beenidentified, along with their association with different types ofcancers. For example, phosphatase and tensin homolog on chromosome ten(PTEN) is among the best-characterized tumor suppressors. PTEN mutationshave been reported in a variety of human cancers¹, including prostatecancer (PCa). Loss of one PTEN allele is seen in the majority oflocalized PCa, and homozygous deletion of PTEN is more common inmetastatic castration-resistant PCa (mCRPC) (˜50%) than inandrogen-dependent primary tumors (˜10%)²⁻⁸. Moreover, loss of PTENprotein expression is more frequent than genomic PTEN loss and has beencorrelated with high Gleason score and faster progression tometastasis⁹. PTEN encodes a dual phosphatase that acts on both lipid andprotein substrates. By catalyzing phosphatidylinositol(3,4,5)-trisphosphate (PIP3) dephosphorylation, PTEN negativelyregulates the phosphatidylinositol 3-kinase (PI3K)-AKT pathway¹⁰⁻¹⁴ akey signaling mediator of most receptor tyrosine kinases (RTKs)¹⁵.Recent integrative genomic profiling and whole-exosome sequencinganalysis highlight the frequency of alterations of the PI3K-AKT pathwayin PCa and are associated with both primary (42%) and metastatic disease(˜100%)^(16, 17). Since activation of the PI3K-AKT pathway upon PTENloss enhances tumor cell survival, proliferation^(12, 18),migration^(19, 20,) angiogenesis^(21, 22), and anti-apoptosis¹⁰,blocking this pathway has been proposed to inhibit tumor growth andsensitize tumor cells to apoptosis.

Reversal of the phenotype induced by loss of tumor suppressors has longproven an elusive goal. Two major strategies have been employed forsuppressor restoration: restoring a functional copy of a giventumor-suppressor gene via transfection; and the use of small-moleculeagents to reactivate tumor-suppressor function via conformational changein the mutated molecule. The latter approach has met with little successand is destined to be ineffective when the suppressor gene has beendeleted. For instance, while pharmacological inhibitors of the AKT-mTORpathway are in clinical development, they cannot compensate fully forthe loss of PTEN function, and show a poor toxicity profile. The majorlimitations of restoring suppressor gene in tumors include inefficientdelivery to targeted tumor cells, poor transfection efficacy,insufficient expression, and possible insertional mutagenesis.Consequently, restoring tumor-suppressor activity in cancer cells ishighly challenging and requires the design of a functionally improvedtumor suppressor with unique therapeutic modality that can withstand therigors of systemic delivery, especially in the metastatic setting wherethe tumor burden is widely distributed.

Recently, chemically modified mRNA has emerged as an intriguingalternative to DNA-based gene therapy, as its intrinsic qualitiesfacilitate its ease of use as a genetic material that is independent ofnuclear localization and genomic integration for transfectionactivity^(23, 24). mRNA also provides rapid protein expression even innon-dividing and hard-to-transfect cells (e.g., immune cells), as wellas cancer cells. Moreover, mRNA offers more consistent and predictableprotein expression kinetics than DNA, whose expression kinetics showrandom onset²⁵⁻²⁸. However, delivery of mRNA presents several potentialchallenges, including large size, highly negative charge, susceptibilityto degradation, and suboptimal protein translation capacity if it is noteffectively modified and delivered into cells²⁹. Thus, safe andeffective in vivo cytosolic delivery of mRNA to tumor tissues whileretaining integrity and functional activity remains elusive.

It is therefore an object of the present invention to providenanotechnologies which can be used to deliver a therapeutic tumorsuppressor mRNA or combinations of different tumor suppressor mRNAs forcancer treatment.

A number of nano-engineered formulations (e.g., polymer-basedNPs^(30, 31), lipid-based NPs³²⁻³s, and lipidoid NPs^(36, 37)) havedesigned for in vivo delivery of RNAs including siRNA or other smalloligonucleotides, and have shown promising results in laboratory orclinical settings³⁸⁻⁴¹. The present methods and compositions areexemplified by the use of a lipid-polymer hybrid NP platform forsystemic delivery of modified PTEN mRNA to PCa tumors. This systemsuccessfully restored functional PTEN protein production, withconsequent inhibition of tumor cell growth and induction of apoptosisboth in vitro and in vivo. As described herein, tumor suppressor mRNAdelivery can be used for the treatment of cancers with a tumorsuppressor deficiency, e.g., hybrid NP-mediated delivery of PTEN mRNA ina cancer with a PTEN deficiency.

Thus, provided herein are compositions comprising one or more tumorsuppressor-encoding mRNAs complexed with a delivery vehicle.

In some embodiments, the delivery vehicle is selected from the groupconsisting of protamine complexes, lipid nanoparticles, polymericnanoparticles, lipid-polymer hybrid nanoparticles, and inorganicnanoparticles, or combinations thereof.

In some embodiments, the delivery vehicle is a lipid-polymernanoparticle. In some embodiments, the core of the nanoparticlecomprises a lipid, a water-insoluble polymer, and the tumorsuppressor-encoding mRNAs are complexed with the lipid. In someembodiments, the lipid comprises cationic lipid-like compound G0-C14. Insome embodiments, the water-insoluble polymer comprises PLGA.

In some embodiments, the tumor suppressor-encoding mRNAs encodesPhosphatase and tensin homolog on chromosome ten (PTEN).

In some embodiments, the tumor suppressor-encoding mRNAs encode one ormore proteins listed in Table 1.

In some embodiments, the tumor suppressor-encoding mRNAs comprise one ormore modifications, preferably selected from the group consisting ofARCA capping; enzymatic polyadenylation to add a tail of 100-250adenosine residues; and substitution of one or both of cytidine with5-methylcytidine and/or uridine with pseudouridine.

Also provided herein are methods for treating a subject who has cancer.The methods include administering to the subject a therapeuticallyeffective amount of a composition described herein.

Further provided herein are methods for treating a subject who hascancer. The methods include administering to the subject atherapeutically effective amount of a composition comprising mRNAencoding Phosphatase and tensin homolog on chromosome ten (PTEN)protein, wherein the mRNAs are complexed with a delivery vehicle, to asubject in need thereof. In some embodiments, the subject has a cancerassociated with loss of PTEN expression or activity. In someembodiments, the subject has prostate cancer, breast cancer, orglioblastoma.

In some embodiments, wherein the delivery vehicle is selected from thegroup consisting of protamine complexes, lipid nanoparticles, polymericnanoparticles, lipid-polymer hybrid nanoparticles, and goldnanoparticles.

In some embodiments, the nanoparticle is a lipid-polymer nanoparticle.

In some embodiments, the core of the nanoparticle comprises a lipid, awater-insoluble polymer, and the tumor suppressor-encoding mRNAs arecomplexed with the lipid. In some embodiments, the lipid comprisescationic lipid-like compound G0-C14. In some embodiments, thewater-insoluble polymer comprises PLGA.

In some embodiments, the tumor suppressor-encoding mRNAs comprise one ormore modifications, preferably selected from the group consisting ofARCA capping; enzymatic polyadenylation to add a tail of 100-250adenosine residues; and substitution of one or both of cytidine with5-methylcytidine and/or uridine with pseudouridine.

Also provided are the compositions as described herein for use intreating a subject who has cancer, e.g., a cancer associated with lossof expression or activity of the tumor suppressor. In some embodiments,the tumor suppressor-encoding mRNAs comprise mRNAs encoding Phosphataseand tensin homolog on chromosome ten (PTEN) protein, and the subject hasa cancer associated with loss of expression or activity of PTEN.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. Other features andadvantages of the invention will be apparent from the following detaileddescription and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-D. Preparation and characterization of mRNA NP. (A)Self-assembly process of polymer-lipid hybrid mRNA NP and schematicrepresentation of NP structure (i-iv). After self-assembly of cationicG0-C14 with anionic mRNA together with PLGA, the formulatedpolymer-lipid hybrid core was coated with lipid-PEG (EGFP-mRNA was usedas reporter mRNA). (B) Agarose gel electrophoresis assay of mRNAstability in organic solvent, naked or complexed with cationic G0-C14,at various weight ratios (from 1˜20). The formulated mRNAPLGA/G0-C14/ceramide-PEG (PGCP) NP was also run through gel to detectany mRNA leaching from the NP. About 0.125 μg of EGFP-mRNA was used forall groups in this assay. (C) The mRNA-PGCP NP was characterized withNanoSIGHT to check size distribution (n=3 batches, 121.5±2.3 nm), andtransmission electron microscopy (TEM) to observe morphology. A weightratio of 1:15 for mRNA: G0-C14 was used for the NP preparation. (D)Stability of mRNA-PGCP NP in 10% serum condition at 37° C. was evaluatedby measuring particle size changes determined with NanoSIGHT at varioustime points up to 48 h (mean±SD, n=3).

FIGS. 2A-F. In vitro toxicity and transfection efficiency of mRNA NPs inPC3 cells. Cells were treated with various mRNA concentrations (at0.062, 0.125, 0.250, 0.500 μg/mL) of EGFP-mRNA-PGCP NPs for 16 h andfurther incubated for 24 h in standard cell culture incubationconditions. (A) AlamarBlue cytotoxicity assay; cell viability wasnormalized with the untreated control group (n=6 per group). (B)Transfection efficiency (% GFP positive cells) was determined using flowcytometry (mean±SD, n=4, ****P<0.0001), and (C) analyzed with thehistograms for the respective groups using Flowjo software. (D)Fluorescence microscopy images of PC3 cells transfected with naked EGFPmRNA, EGFP-mRNA-PGCP NP, and Lipofectamine 2000 (L2K)-EGFP-mRNA(magnification at 20×). (E) Mechanism of cellular uptake and endosomalescape of mRNA NPs in PC3 cells. The cells were pre-incubated for 30 minin serum-free medium containing inhibitors (Filipin, CPZ, EIPA, and BafA1 were used as the inhibitor for caveolae-mediated endocytosis,clathrin-mediated endocytosis, macropinocytosis, and intracellularproton pump effects, respectively) or combinations of inhibitors(Filipin, CPZ, or EIPA mixed with Baf A1) prior to transfection withEGFP-mRNA PGCP NPs at mRNA concentration of 0.250 μg/ml. Transfectionefficiency (% GFP positive cells) was determined using flow cytometry(mean±SD, n=5, ***P<0.001 and ****P<0.0001), and (F) the mechanism ofcellular uptake and intracellular transport of the hybrid mRNA NPs isschematically illustrated.

FIGS. 3A-D. In vitro mechanism of PTEN-mRNA-PGCP NP treatment in PC3cells and its therapeutic effect. (A) Immunofluorescence staining ofHA-PTEN after empty PTEN NP and PTEN-mRNA-PGCP NP treatment. (B) Westernblot analysis of PI3K-AKT pathway signaling after mRNA-PGCP NP treatmentin the presence (+) or absence (−) of serum. (C) The percent cellviability of PC3 cells after treatment with empty PGCP NP,EGFP-mRNA-PGCP NP, or PTEN-mRNA-PGCP NP measured by MTT assay (mean±SD,n=3, **P<0.01 and ***P<0.001). (D) Apoptosis was determined by flowcytometry after empty PGCP NP, EGFP-mRNA-PGCP NP, or PTEN-mRNA-PGCP NPtreatment of PC3 cells.

FIGS. 4A-B. Effect of lipid-PEG on pharmacokinetics and biodistributionof mRNA NP. (A) Circulation profile of naked Cy5 EGFP mRNA, and twodifferent mRNA NP formulations with ceramide-PEG (termed asCy5-EGFP-mRNA-PGCP NP) and DSPE-PEG (termed as Cy5-EGFP-mRNA-PGDP NP) innormal Balb/c male mice after injection (i.v., tail vein) (n=3,mean±SEM). (B) Biodistribution of naked Cy5 EGFP mRNA,Cy5-EGFP-mRNA-PGCP NP, and Cy5-EGFP-mRNA-PGDP NP in different organsincluding tumors in athymic nude mice bearing PC3-xenograft tumor 24 hpost-injection.

FIG. 5A-E. In vivo therapeutic validation of PTEN restoration usingPTEN-mRNA NP in PCa xenograft model. (A) Scheme of tumor inoculation andsystemic injection (i.v., tail-vein) of PBS, EGFP-mRNA-PGDP NP, orPTEN-mRNA-PGDP NP in PC3 tumor-bearing male athymic nude mice. Mice wereinjected at day 10 post tumor inoculation. Injections were performedevery three days for 6 times. (B) Whole-body images of mice bearingPC3-xenograft tumors treated with PBS, EGFP-mRNA-PGDP NP, andPTEN-mRNA-PGDP NP (at day 35). (C) Tumor growth to show in vivotherapeutic efficacy of PTEN-mRNA-PGDP NP (n=8) as compared to PBS (n=7)and EGFP-mRNA-PGDP NP (n=9) (mean±SEM; *P<0.05 vs. PBS or EGFP-mRNA-PGDPNP. The arrows indicate i.v., tail vein injections. Tumor sizemeasurement began on day 10 and continued every three days until day 43.The representative excised tumor images are also shown on the right. (D)The average body weight of the PC3 tumor-bearing xenograft mice over thecourse of therapy (mean±SEM). The effect of PTEN-mRNA-PGDP NP treatmentin PC3 tumor-bearing xenograft mice was evaluated by (E)Immunohistochemistry staining of HA-PTEN (bar indicates 200 μm) on fixedtumor tissue after PBS, EGFP-mRNA-PGDP NP, or PTEN-mRNA-PGDP NPtreatment (scale bar: 200 μm).

FIGS. 6A-C In vivo therapeutic validation of PTEN restoration usingPTEN-mRNA NPs in disseminated metastatic PCa model. (A) Scheme of i.v.tumor inoculation and systemic injection (i.v., tail-vein) of PBS,EGFP-mRNA-PGDP NP, or PTEN-mRNA-PGDP NP (n=8 mice per cohort) indisseminated PC3-luc metastatic male athymic nude mice. (B)Bioluminescent imaging for disseminated PC3-luc metastatic tumors atdifferent time points post treatment. (C) Fold change in averageradiance per mouse by normalizing to day 0 tumor burden as determined bybioluminescent imaging; inset: fold change in average radiance per mouseat experimental endpoint (day 15) for each treatment group (mean±SEM).

FIGS. 7A-C. In vivo therapeutic validation of PTEN restoration usingPTEN-mRNA NPs in IT orthotopic PCa model. (A) Scheme of IT tumorinoculation and systemic injection (i.v., tail-vein) of PBS,EGFP-mRNA-PGDP NP, or PTEN-mRNA-PGDP NP (n=12 mice; n=24 tibiae percohort) in IT PC3-luc-bearing male athymic nude mice. (B) Bioluminescentimaging of total radiance of PC3-luc at time points post IT injectionsand treatments. (C) Fold change in average radiance per tibia asnormalized to day 0 tumor burden as determined by bioluminescentimaging; inset: fold change in average radiance per tibia atexperimental endpoint (day 15) for each treatment group (mean±SEM).

FIGS. 8A-B. In vivo toxicity studies by histopathological andhematological analysis after treatment of mRNA NPs vs. PBS. Forhistopathological assay, H&E staining of tissue sections of major organswas analyzed three days after the last injection of PBS, EGFP-mRNA-PGDPNP, and PTEN-mRNA-PGDP NP at day 28 (A), and at the endpoint of day 43(b) post tumor inoculation as shown in the scheme of FIG. 6A. Imageswere taken at 20× magnification. For hematological assay, the levels ofaspartate aminotransferase (AST), alanine aminotransferase (ALT), bloodurea nitrogen (BUN), creatinine, and troponin-1 in serum were measuredat day 28 (a) and at day 43 (b) post tumor inoculation (mean±SD, n=5,n.d.: not detectable).

FIGS. 9A-C. In vitro toxicity and transfection efficiency of mRNA NP inDU145 cells, which were treated with various mRNA concentrations (at0.062, 0.125, 0.250, 0.500 μg/mL) of EGFP-mRNA-PGCP NP. (A) AlamarBluecytotoxicity assay. Percent cell viability was normalized with theuntreated control group (mean±SD, n=3). (B) Transfection efficiencypercentages were determined using flow cytometry (mean±SD, n=4) and (C)the histograms for the respective groups were generated after analysisusing Flowjo software.

FIGS. 10A-C In vitro toxicity and transfection efficiency of mRNA NP inLNCaP cells. The cells were treated with various mRNA concentrations (at0.062, 0.125, 0.250, 0.500 μg/mL) of EGFP-mRNA-PGCP NP. (A) AlamarBluecytotoxicity assay. The cell viability percentages were normalized withthe untreated control group (mean±SD, n=3). (B) Transfection efficiencypercentages were determined using flow cytometry (mean±SD, n=4,***P<0.001), and (C) the histograms for the respective groups weregenerated after analysis using Flowjo software.

FIGS. 11A-B. The effect of RNase on the activity of mRNA NPs. (a)Transfection efficiency percentages of naked mRNA (complexed with L2K)and EGFP-mRNA-PGCP NPs in the presence of two RNase concentrations (1mg/ml and 10 mg/ml) were determined using flow cytometry (mean±SD, n=4,****P<0.0001). (b) The histograms for the respective groups weregenerated after analysis using Flowjo software.

FIGS. 12A-C. The effect of PTEN-mRNA in PC3 cells transfected byLipofectamine 2000 (L2K). (A) Immunofluorescence staining of HA-PTENexpression in cells after transfection with PTEN mRNA or pHAGE-PTEN WT.(B) Downregulation of PI3K-AKT pathway after treatment with PTEN mRNA.(C) Cell viability of PC3 cells after PTEN mRNA treatment determined byCyQUANT assay (mean±SD, n=3, **P<0.01 and ***P<0.001).

FIGS. 13A-B. In vitro therapeutic effect of PTEN-mRNA-PGCP NP in LNCaPprostate cancer cells. (A) Cell viability of LNCaP and its invasivesubclone LNCaP LN3 cells after empty PGCP NP, EGFP-mRNA-PGCP NP, orPTEN-mRNA-PGCP NP treatment, measured by MTT assay (mean±SD, n=3,***P<0.001). (B) Apoptosis assay of LNCaP cells after empty PGCP NP,EGFP-mRNA-PGCP, or PTEN-mRNA-PGCP NP treatment.

FIGS. 14A-B. In vitro therapeutic effect of PTEN-mRNA-PGCP NP inprostate epithelial cells (PreC) and DU145 prostate cancer cells. (A)Cell viability of PreC and DU145 cells after empty PGCP NP,EGFP-mRNA-PGCP, or PTEN-mRNA-PGCP NP treatment measured by MTT assay.(B) Western blot analysis of PI3K-AKT pathway in DU145 cells underdifferent PGCP NP treatment conditions.

FIGS. 15A-C. In vitro therapeutic validation of PTEN-mRNA-PGCP NP inMDA-MB-468 and MDA-MB-231 breast cancer cells. (A) Cell viability ofMDA-MB-468 and MDA-MB-231 cells after empty PGCP NP, EGFP-mRNA-PGCP NP,or PTEN-mRNA-PGCP NP treatment measured by MTT assay (mean±SD, n=3,***P<0.001). (B) Western blot analysis of PI3K-AKT pathway signaling,and (C) apoptosis assay of MDA-MB-468 cells after empty, EGFP-mRNA, orPTEN-mRNA-PGCP NP treatment.

FIGS. 16A-B. Effect of DSPE-PEG on mRNA NP formulation. (A) ThemRNA-PGDP NP (prepared with DSPE-PEG) was characterized with NanoSIGHTto check size distribution (112.7±1.3 nm), transmission electronmicroscopy (TEM) to observe morphology, and dynamic light scattering(DLS) to measure surface charge (5.22±0.43 mV). A weight ratio of 1:15for mRNA: G0-C14 was the best formulation. (B) Stability ofEGFP-mRNA-PGDP NP in 10% serum condition was evaluated by measuringparticle size changes determined by NanoSIGHT at various time periodsuntil 48 h.

FIG. 17. In vitro transfection efficiency of EGFP-mRNA-PGDP NP in PC3cells. Cells were treated with various mRNA concentrations (at 0.062,0.125, 0.250, 0.500 μg/ml) of EGFP-mRNA-PGDP NP for 16 h and furtherincubated for 24 h under standard cell-culture incubation conditions.Transfection efficiency (% GFP positive cells) was determined using flowcytometry (mean±SD, n=4) and analyzed with the histograms for therespective groups using Flowjo software.

FIGS. 18A-B. Biodistribution of Cy5-PTEN-mRNA NP. (A) Accumulation ofnaked Cy5-PTEN-mRNA, Cy5-PTEN-mRNA-PGCP NP, and Cy5-PTEN-mRNA-PGDP NP indifferent organs including tumors in athymic nude mice bearingPC3-xenograft tumor 24 h post-injection. (B) Mean fluorescent intensity(MFI) was analyzed using ImageJ software and graphed using GraphPadPrism (mean±SD, n=3).

FIG. 18C. The average tumor weight of the PC3 tumor-bearing xenograftmice over the course of therapy with PBS (n=5), EGFP-mRNA-PGDP NP (n=6),and PTEN-mRNA-PGDP NP (n=6) (mean±SEM, *P<0.05 vs. EGFP-mRNA-PGDP NP and** P<0.01 vs. PBS).

FIGS. 19A-B. In vivo therapeutic validation of PTEN restoration usingPTEN-mRNA NPs in advanced PCa (PC3-disseminated metastatic and ITorthotopic) models. (A) Fold change in average radiance per mouse asnormalized to day 0 tumor burden determined by bioluminescent imaging,represented as a waterfall plot with each bar representing one mouse(n=8 mice per cohort). (B) Fold change in average radiance per tibiapost IT injection of PC3-luc cells as normalized to day 0 tumor burdendetermined by bioluminescent imaging, represented as a waterfall plotwith each bar representing one tibia (n=12 mice; n=24 tibae per cohort).

FIGS. 20A-B. The average mouse body weight of the (A) PC3-lucdisseminated metastatic mice (n=8 mice per cohort), and (B) ITorthotopic PC3-luc-bearing mice (n=12 mice per cohort), over the courseof therapy (mean±SEM).

FIG. 21. In vivo toxicity assessment by measuring immune stimulationafter treatment of immunocompetent BALB/c male normal mice with PBS,naked PTEN mRNA, empty PGDP NP, and PTEN-mRNA-PGDP NP. Serum levels ofTNF-α were measured 6 and 24 h post injection (i.v., tail vein) of theabove groups (n=3, mean±SEM).

DETAILED DESCRIPTION

Loss and/or mutation of tumor-suppressor genes is a dominant force intumor development and clinical resistance to a variety of therapies⁵³.Reversal of the phenotype induced by loss of tumor suppressors has longproven an elusive goal. Two major strategies have been employed forsuppressor restoration: restoring a functional copy of a giventumor-suppressor gene via transfection; and the use of small-moleculeagents to reactivate tumor-suppressor function via conformational changein the mutated molecule⁵⁴. Major hurdles in restoring suppressor gene intumors have included inefficient delivery to targeted tumor cells, poortransfection efficacy, insufficient expression, and possible insertionalmutagenesis. Consequently, restoring tumor-suppressor activity in cancercells is highly challenging and requires the design of a functionallyimproved tumor suppressor with unique therapeutic modality that canwithstand the rigors of systemic delivery, especially in the metastaticsetting where the tumor burden is widely distributed.

The tumor suppressor-mRNA delivery platform reported herein is anexample of such an approach. To the best of the present inventors'knowledge, this is the first report of any kind of tumor inhibitionfollowing direct systemic restoration of tumor-suppressor gene using anmRNA delivery strategy in vivo. Use of an mRNA delivery platform allowsrapid gene expression with controlled and predictable expressionkinetics, higher transfection efficiency, and (most importantly) caneliminate genomic complexation and mutagenesis due to the inherentcytoplasmic and diminished nuclear expression of the desired protein²⁹.In addition, described herein is a delivery system that in someembodiments uses a new-generation lipid-polymer hybrid NP platform thatprovides effective intravenous delivery of the suppressor mRNA to tumorxenografts. The end result is restoration of function of the exemplarytumor suppressor PTEN as illustrated by inhibited both primary andadvanced tumor growth, increased apoptosis, and blockade of the PI3K-AKTsignaling pathway. Because PTEN loss is frequent in late-stage PCa, thisapproach can be useful in this patient population.

In previous reports, the most widely investigated non-viral genedelivery carriers such as polyethylenimine (PEI), DOTAP/cholesterolliposome, and the DOTAP/DOPE system^(49, 55-57) provided only suboptimaland variable mRNA transfection efficacy (40-80%) in cancer cells andfibroblasts (e.g., HeLa and NIH 3T3 cells). In addition, in PCa cells(e.g., PC3) it was reported that mRNA transfection efficacy of thePEI/mRNA complex was only 30%, although those researchers found a highin vitro mRNA transfection activity in PC3 cells using a histidine-richreducible polycation system⁵⁸. The exemplary polymer-lipid hybrid mRNANPs described herein, which were prepared using a robust self-assemblystrategy, provided 86-98.2% mRNA transfection capacity with minimaltoxicity in various PCa cell lines (e.g., LNCaP, PC3, and DU145), a newstandard for in vitro delivery of mRNA to tumors. Potential reasons forthis effective mRNA delivery to tumors may be the relatively small andtunable size of the NPs (about 20-250 nm), as well as their high andtunable mRNA encapsulation efficiency (10-100%) and loading efficiency(0.2-20%), compact shape, and good stability. Whereas smaller particlesize could be achieved with small oligonucleotides such as siRNA, it ismore difficult with larger payloads such as mRNA. It is worth notingthat such small NPs may more efficiently permeate the leaky tumormicrovasculature and achieve greater tumor accumulation and deepertissue penetration compared to larger NPs⁵⁹⁻⁶¹.

For systemic in vivo application, PEGylation of NPs is a well-documentedstrategy to prevent rapid elimination from the circulation, since itreduces the interaction between NPs and serum proteins followingrecognition by mononuclear phagocytic system-mediated clearancemechanisms^(62,63). Simultaneously, PEGylation could also hinder NPinteractions with the target cell membrane, which may decrease tumorcell-mediated uptake. Therefore, proper dissociation of lipid-PEGmolecules is necessary for optimal systemic circulation andextravasation of our mRNA NPs at the tumor site as well as effectiveuptake by tumor cells. The dissociation kinetics depend on the structureof the lipid-PEG molecules in the hybrid NPs and may be controlled bythe length and/or saturation of lipophilic tails⁴⁵. In that context,DSPE-PEG exhibited relatively slow de-PEGylation profile compared toceramide-PEG, as described earlier from measurements of lipid-PEG'sdissociation kinetics from NPs in the presence of serum albumin, whichis the most abundant plasma protein and binds with diacyl lipids⁶⁴.Moreover, a quicker release of ceramide-PEG than DSPE-PEG from NPs wasobserved, and the surface charge of the NPs changed over time afterlipid-PEG dissociation: the charge of ceramide-PEG NPs rapidly increasedfrom 2.2 to 31.4 mV in 3 h, but slowly for DSPE-PEG NPs from −4.0 to11.9 mV over 24 h, although both NPs were initially near neutral. Thisslow de-PEGylation profile conferred better PK, tumor biodistribution,and therapeutic efficacy for DSPE-PEG NPs⁴⁵. Thus the hybrid mRNA NPcoated with DSPE-PEG showed higher stability, longer circulation, andincreased tumor accumulation compared to ceramide-PEG NP, indicatingmore efficient systemic restoration of mRNA to tumors and implicationsbeyond any particular tumor suppressor and cancer type.

PTEN loss has been recognized for two decades as being involved in PCaprogression⁶⁵⁻⁶⁷; surprisingly, there has been no progress on directrestoration of PTEN function in PTEN-null PCa cells, presumably due tothe inefficient delivery and insufficient expression of PTEN at thetumor site. Recently, a secreted form of PTEN called PTEN-long (PTEN-L)that can penetrate cells has been discovered and shown to rescue PTENfunction in PTEN-null U87-MG glioblastoma cells and MDA-MB-468 breastcancer cells in vitro and in vivo⁶⁸.

The present approach is generalizable to a wide variety oftumor-suppressive molecules. Recent protein structure studies havereported that PTEN and PTEN-L have different properties relating toconformational change, membrane binding, and substrate specificity⁶⁹.Future studies will be necessary to explore the relative efficacy ofthese two approaches. It is further likely that delivery of mRNA NPswill achieve greater tumor-specific distribution than free protein. Itwas also recently reported that PTEN loss promotes resistance toclinical therapy as well as T-cell-mediated immunotherapy^(70, 71), thusrestoring functional PTEN via the PTEN mRNA NP platform described hereincan also be used in combination with immunotherapeutic applications andin rescuing chemosensitivity in resistant tumors; delivery of mRNA NPsalso holds the potential to potentiate other therapeutic approaches²⁹.The present findings provide significant support for theproof-of-concept that the systemic restoration of PTEN rescues PTENfunction in PTEN-null PCa and effectively suppresses tumor progressionwith negligible side-effects.

Thus, provided herein are methods for NP-mediated mRNA delivery of tumorsuppressors (e.g., PTEN) as therapeutics for the treatment of cancer.

Methods of Treatment

The methods described herein include methods for the treatment ofcancers associated with loss of a tumor suppressor. In some embodiments,the disorder is PTEN-null Prostate Cancer (PCa). Generally, the methodsinclude administering a therapeutically effective amount of one or moretumor suppressor mRNAs complexed with a delivery vehicle as describedherein, e.g., a nanoparticle, to a subject who is in need of, or who hasbeen determined to be in need of, such treatment. In some embodiments,the delivery vehicle (e.g., nanoparticle) is complexed with mRNAs thatencode a single tumor suppressor; in other embodiments, the vehicle iscomplexed with mRNAs coding for multiple tumor suppressors. In someembodiments, the methods include administering a plurality vehicle-RNAcomplexes that include vehicles complexed with two or more mRNAs, e.g.,wherein the vehicles are each complexed with only a single kind of mRNA(i.e., each vehicle is complexed with mRNA encoding one tumorsuppressor), or wherein the vehicles are each complexed with two or morekinds of mRNAs (i.e., the vehicles are each complexed with mRNAsencoding two or more tumor suppressors).

As used in this context, to “treat” means to ameliorate at least onesymptom of the cancer associated with loss of a tumor suppressor.Administration of a therapeutically effective amount of a compounddescribed herein for the treatment of a cancer associated with loss of atumor suppressor can result in, for example, increased expression of thetumor suppressor, and one or more of reduced tumor size, reduced tumorgrowth rate, reduced risk of metastasis, decrease risk of reoccurrence,and reduced number of tumors.

As used herein, the terms “cancer” refers to cells having the capacityfor autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. The term is meant toinclude all types of cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues, or organs,irrespective of histopathologic type or stage of invasiveness.“Pathologic hyperproliferative” cells occur in disease statescharacterized by malignant tumor growth.

The terms “cancer” or “neoplasms” include malignancies of the variousorgan systems, such as affecting lung, breast, thyroid, lymphoid,gastrointestinal, and genito-urinary tract, as well as adenocarcinomasthat include malignancies such as most colon cancers, renal-cellcarcinoma, prostate cancer and/or testicular tumors, non-small cellcarcinoma of the lung, cancer of the small intestine and cancer of theesophagus.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. In some embodiments, thedisease is renal carcinoma or melanoma. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

The term “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

In some embodiments, the methods also include administering one or moreimmunotherapies.

Tumor Suppressor mRNA

The present methods include delivering mRNA encoding a tumor suppressorto a cell (e.g., a tumor cell) lacking that tumor suppressor. As usedherein, a tumor suppressor is a protein that acts to reduce thepotential for cancer and tumor formation by modulating cell growth, bynegative regulation of the cell cycle or by promoting apoptosis. Thus,loss of a tumor suppressor (e.g., through mutation or dysregulation) canlead to unregulated cell growth and tumor development. Mutations andother alterations that are associated with cancer for each of the aboveare known in the art.

A number of Tumor Suppressors are known in the art. See, e.g., Table 1.

TABLE 1 Genetic Associated GenBank Acc No. GENE Alteration(s) Cancer(s)mRNA Protein PTEN Point Prostate, breast, AF067844.1 AAD13528.1mutation, glioblastoma, deletion melanoma, pancreatic cancer, colorectalcancer, leukemia APC Point Adenomatous M74088.1 AAA03586.1 mutation,polyposis and deletion sporadic colorectal tumors, gastric cancer ARFDeletion Breast carcinomas, AF208864.1 AAF64278.1 colorectal adenoma,glioblastoma BMPR Point mutation Gastrointestinal NM_009758.4NP_033888.2 cancer BRCA1 Point mutation Ductal breast U14680.1AAA73985.1 cancers, Epithelial ovarian cancers E-cadherin Point mutationLoss of function Z13009.1 CAA78353.1 leads to metastasis EXT1,2 PointHereditary multiple S79639.1, AAB62283.1 mutation, exostoses, alsoU62740.1 AAB07008.1 deletion, known as diaphyseal insertion aclasisFBXW7 Point Breast cancer AF411971.1 AAL06290.1 mutation, deletion FHPoint mutation Hereditary BC003108.1 AAH03108.1 leiomyomatosis andrenal-cell cancer GPC3 Deletions, Lung carcinoma L47125.1 AAA98132.1point mutation HIPK2 Point mutation Metastatic bladder AF208291AAG41236.1 cancer HRPT2 Point mutation Hereditary NM_024529.4NP_078805.3 (CDC73) hyperparathyroidism- jaw tumor syndrome, Malignancyin sporadic parathyroid tumors INPP4B Deletion, loss Epithelial U96922.1AAB72153.1 of carcinomas and some heterozygosity, human basal-likereduced breast carcinomas expression LKB1 Point Human Lung CancerU63333.1 AAB05809.1 mutation, (especially NSCLC), deletion cervicalcarcinomas Inherited cancer disorder Peutz- Jeghers Syndrome MEN1 Pointmutation Pituitary tumors U93236.1 AAC51228.1 MMR Point Hereditary non-NM_002438.3 NP_002429.1 (MRC1) mutation, polyposis colon reduced cancerexpression MUTYH Point Lung and ovarian U63329.1 AAC50618.1 mutation,tumors, and deletion lymphomas NF1 Point Juvenile NM_000267.3NP_000258.1 mutation, myelomonocytic deletion leukemia, Watson syndromeand breast cancer. NF2 Point Meningioma L11353.1 AAA36212.1 mutation,Thyroid cancer, deletion mesothelioma, and melanoma p15, Point mutationColorectal cancer, AB060808.1 BAB91133.1 p16 leukemia L27211.1AAA92554.1 p53 Point Lung AF307851.1 AAG28785.1 mutation, ProstateNM_000546.5 NP_000537.3 deletion p57 Point mutation Beckwith-NM_000076.2 NP_000067.1 (CDKN1C) Wiedemann syndrome Ptch Point mutationCell carcinomas of NM_000264.4 NP_000255.2 the skin, ovarian fibromas,and medulloblastomas RB1 Point Prostate cancer NM_000321.2 NP_000312.2mutation, Pituitary deletion melanotroph tumors RECQL4 Point mutationOsteosarcoma AB006532.1 BAA74453.1 SDH Point Paraganglioma, renalU17248.1 AAA81167.1 mutation, cell carcinoma deletion Smad2/3 PointBreast cancer U65019.1 AAB17054.1 mutation, BC050743.1 AAH50743.1deletions Smad4 Point mutation Pancreatic U44378.1 AAA91041.1 GastricCarcinoma Su(Fu) Point Brain tumor NM_016169.3 NP_057253.2 mutation,deletion TGFβR Point mutation Head and neck NM_001306210.1NP_001293139.1 cancers, cervical and ovarian carcinomas TSC1/TSC2 Pointmutation Tuberous sclerosis AF013168.1 AAC51674.1 complex AB014460.1BAA32694.1 VHL Point Renal carcinomas NM_000551.3 NP_000542.1 mutation,deletion, hyper- methylation WT1 Point Haematological NM_000378.4NP_000369.3 mutation, malignancies deletion Pediatric nephroblastomaWilms tumor XPA Point mutation Bladder cancer NM_000380.3 NP_000371.1XPC Point ESCC, gastric cancer NM_004628.4 NP_004619.3 mutations, splicevariants XPD Point mutation Glioma, NSCLC, NM_000400.3 NP_000391.1(ERCC2) Sarcoma α-catenin Point mutation Basal-like breastNM_001323983.1 NP_001310912.1 (CTNNA1) cancer RASSF1A Hyper- Lung,Cervical NM_007182.4 NP_009113.3 methylation, Cancer point mutation SDHBPoint mutation Kidney NM_003000.2 NP_002991.2 Paragangliomas SIN3B Pointmutation Prostate cancer NM_015260.3 NP_056075.1 RGS12 Point mutationProstate cancer NM_198227.1 NP_937870.1 Kail Deletion, Prostate cancerNM_002231.3 NP_002222.1 metastasis mutation and suppressor loss of(CD82) expression ING1B Point mutation Prostate cancer, NM_198218.2NP_937861.1 Brain tumors Atg7 Deletion Prostate cancer NM_001349232.1NP_001336161.1 JARID1D Point mutation Prostate cancer NM_001146705.1NP_001140177.1 (KDM5D) PALB2 Point mutation Breast cancer NM_024675.3NP_078951.2 TP53BP1 Point mutation Breast cancer NM_001141980.2NP_001135452.1 RAD51 Point mutation Breast cancer NM_133487.3NP_597994.3 XRCC4 Point mutation Breast cancer NM_003401.4 NP_003392.1KEAP1 Point mutation Liver cancer NM_203500.1 NP_987096.1 KEAP1 Pointmutation Liver cancer NM_012289.3 NP_036421.2 RPS6KA3 Point mutationLiver cancer NM_004586.2 NP_004577.1 RARβ Point mutation Lung cancerNM_000965.4 NP_000956.2 FHIT Point mutation Lung cancer NM_002012.3NP_002003.1 PTCH1 Point mutation Lung cancer NM_001083602.2NP_001077071.1 DCC Point mutation Colorectal cancer NM_005215.3NP_005206.2 BAX Point mutation Colorectal cancer NM_001291428.1NP_001278357.1 AML1 Point mutation Acute myeloid NM_001754.4 NP_001745.2(RUNX1) leukemia CDKN2A Point mutation Bladder NM_000077.4 NP_000068.1CDKN1B Point mutation Prostate cancer NM_004064.4 NP_004055.1 NKX3-1Point mutation Prostate cancer NM_006167.3 NP_006158.2 RPP14 Pointmutation Melanoma NM_001098783.2 NP_001092253.1 CDK4 Point mutationMelanoma NM_000075.3 NP_000066.1 CDK6 Point mutation MelanomaNM_001259.7 NP_001250.1The above sequences are exemplary, as some of the above genes may havemultiple transcript variants; generally speaking, the methods caninclude using an mRNA sequence for the variant that is predominantlyexpressed in a normal, non-cancerous cell of the same type as the tumor.The methods can include using a nucleotide sequence coding for an mRNAthat is at least 80% identical to a reference sequence in Table 1. Insome embodiments, the nucleotide sequences are at least 85%, 90%, 95%,99% or 100% identical.

To determine the percent identity of two sequences, the sequences arealigned for optimal comparison purposes (gaps are introduced in one orboth of a first and a second amino acid or nucleic acid sequence asrequired for optimal alignment, and non-homologous sequences can bedisregarded for comparison purposes). The length of a reference sequencealigned for comparison purposes is at least 80% (in some embodiments,about 85%, 90%, 95%, or 100%) of the length of the reference sequence.The nucleotides or residues at corresponding positions are thencompared. When a position in the first sequence is occupied by the samenucleotide or residue as the corresponding position in the secondsequence, then the molecules are identical at that position. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two amino acidsequences can be determined using the Needleman and Wunsch ((1970) J.Mol. Biol. 48:444-453) algorithm which has been incorporated into theGAP program in the GCG software package, using a Blossum 62 scoringmatrix with a gap penalty of 12, a gap extend penalty of 4, and aframeshift gap penalty of 5.

As noted above, the delivery vehicle (e.g., nanoparticle) can becomplexed with one, two or more mRNAs (e.g., a plurality of mRNAs) thatencode a single tumor suppressor, or encoding multiple tumorsuppressors.

In some embodiments, e.g., wherein the cancer is prostate cancer, themRNA is PTEN. In some embodiments, the mRNA is p53. In some embodiments,the mRNA is RB. In some embodiments, the mRNAs are PTEN and p53. In someembodiments, the mRNAs are PTEN and RB. In some embodiments, the mRNAsare RB and p53. In some embodiments, the mRNAs are PTEN, p53 and RB.

In preferred embodiments, the mRNA encodes the human PTEN tumorsuppressor, and in some embodiments, the cancer is breast cancer,prostate cancer, or glioblastoma. In some embodiments, the methodsinclude determining that a subject has a cancer that is associated withloss of a tumor suppressor, and then delivering an mRNA encoding thattumor suppressor to the subject, e.g., to the tumor in the subject.Determining that a subject has a cancer that is associated with loss ofa tumor suppressor can be done using any method known in the art, e.g.,obtaining a sample comprising tumor cells, and detecting the presence ofa mutation or loss of a tumor suppressor in the cells, e.g., bysequencing DNA of the tumor cells and detecting a mutation that is knownto be associated with oncogenesis, or by detecting a decreased level oractivity of the tumor suppressor protein as compared to a reference,e.g., a reference that represents a level or activity of the protein ina normal, non-cancerous cell of the same type as the tumor cell (i.e., acell from the same kind of tissue, a non-cancerous part of the sametissues in the same individual or in an individual who doesn't havecancer).

A mature mRNA is generally comprised of five distinct portions (see FIG.1a of Islam et al., Biomater Sci. 2015 December; 3(12):1519-33): (i) acap structure, (ii) a 5′ untranslated region (5′ UTR), (iii) an openreading frame (ORF), (iv) a 3′ untranslated region (3′ UTR) and (v) apoly(A) tail (a tail of 100-250 adenosine residues). Typically, the mRNAwill be in vitro transcribed using methods known in the art. The mRNAwill typically be modified, e.g., to extend half-life or to reduceimmunogenicity. For example, the mRNA can be capped with an anti-reversecap analog (ARCA), in which OCH₃ is used to replace or remove natural 3′OH cap groups to avoid inappropriate cap orientation. TetraphosphateARCAs or phosphorothioate ARCAs can also be used (Islam et al. 2015).The mRNA is preferably enzymatically polyadenylated (addition of a polyadenine (A) tail to the 3′ end of mRNA), e.g., to comprise a poly-A tailof at least 100 or 150 As. Typically poly(A) polymerase is used; E. colipoly(A) polymerase (E-PAP) I has been optimized to add a poly(A) tail ofat least 150 adenines to the 3′ terminal of in vitro transcribed mRNA.Preferably, any adenylate-uridylate rice elements (AREs) are removed orreplaced with 3′ UTR of a stable mRNA species such as β-globin mRNA.Iron responsive elements (IREs) can be added in the 5′ or 3′ UTR. Insome embodiments, the mRNAs include full or partial (e.g., at least 50%,60%, 70%, 80%, or 90%) substitution of cytidine triphosphate and uridinetriphosphate with naturally occurring 5-methylcytidine and pseudouridine(ψ) triphosphate. See Islam et al., 2015, and references cited therein.

mRNA Delivery Vehicles

In the present methods and compositions, the mRNA encoding a tumorsuppressor is complexed with a delivery vehicle. The delivery vehiclecan include, inter alia, protamine complexes and particles such as lipidnanoparticles, polymeric nanoparticles, lipid-polymer hybridnanoparticles, and inorganic (e.g., gold) nanoparticles, e.g., asdescribed in Islam et al., 2015.

Particles may be microparticles or nanoparticles. Nanoparticles arepreferred for intertissue application, penetration of cells, and certainroutes of administration. The nanoparticles may have any desired sizefor the intended use. The nanoparticles may have any diameter from 10 nmto 1,000 nm. The nanoparticle can have a diameter from 10 nm to 900 nm,from 10 nm to 800 nm, from 10 nm to 700 nm, from 10 nm to 600 nm, from10 nm to 500 nm, from 20 nm from 500 nm, from 30 nm to 500 nm, from 40nm to 500 nm, from 50 nm to 500 nm, from 50 nm to 400 nm, from 50 nm to350 nm, from 50 nm to 300 nm, or from 50 nm to 200 nm. In preferredembodiments the nanoparticles can have a diameter less than 400 nm, lessthan 300 nm, or less than 200 nm. The preferred range is between 50 nmand 300 nm.

A. Nanoparticle as Delivery Vehicles

Nanoparticles can be polymeric particles, non-polymeric particles (e.g.,a metal particle, quantum dot, ceramic, inorganic material, bone, etc.),liposomes, micelles, polymeric micelles, viral particles, hybridsthereof, and/or combinations thereof. In some embodiments, thenanoparticles are, but not limited to, one or a plurality of lipid-basednanoparticles, polymeric nanoparticles, metallic nanoparticles,surfactant-based emulsions, dendrimers, buckyballs, nanowires,virus-like particles, peptide or protein-based particles (such asalbumin nanoparticles) and/or nanoparticles that are developed using acombination of nanomaterials such as lipid-polymer nanoparticles. Insome embodiments, nanoparticles can comprise one or more polymers.

Nanoparticles may be a variety of different shapes, including but notlimited to spheroidal, cubic, pyramidal, oblong, cylindrical, toroidal,and the like. Nanoparticles can comprise one or more surfaces. Exemplarynanoparticles that can be adapted for use include (1) the biodegradablenanoparticles disclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2)the polymeric nanoparticles of Published US Patent Application20060002852 to Saltzman et al., or (4) the lithographically constructednanoparticles of Published US Patent Application 20090028910 to DeSimoneet al.

Additional delivery forms include biodegradable, or non-biodegradablepolymeric units in a form of an implant such as a rod, disc (wafer), ormicrochip, or in particulate form, such as microparticle or nanoparticleform. The delivery forms typically have dimensions suitable forimplantation into tissues. These local delivery forms are placed at adesired site in a subject's body and release the agent(s) locally in adosage not sufficient to cause systemic efficacy or side effects. Forexample, discs may have a diameter of between 1 mm and 10 mm and athickness of between 0.5 mm and 3 mm. The rods may have a length ofbetween 1 mm and 10 mm, and a width of between 0.5 mm and 3 mm.

Polymeric microchips for multi-dose delivery are described by Richards,et al., Nat Mater. (2003) 2(11):709-10 and Kim, et al. J ControlRelease. (2007) 123(2):172-8. Biodegradable polymeric microchips can befabricated as described in these studies for release of the nucleicacids over an extended period, for example, 142 day. As described inthese papers, the microchips were 1.2 cm in diameter, 480-560 micromthick and had 36 reservoirs that could each be filled with a differentchemical. The devices were fabricated from poly(L-lactic acid) and hadpoly(D,L-lactic-co-glycolic acid) membranes of different molecularmasses covering the reservoirs.

A drug delivery system can be designed to release pulses of differentdrugs at intervals after implantation in a patient by using differentmolecular masses or materials for the membrane. The devices can also bedesigned to have differential degradation rates in vivo and in vitro,using different polymer composition and/or molecular weights, such asbiocompatible poly(lactic acid) and poly(glycolic acid) homo- andco-polymers for a polymeric drug-delivery microchip. See U.S. Pat. Nos.6,491,666, 6,527,762, 6,976,982, 7,226,442, and 7,604,628. Suitabledevices can be obtained from Microchips Biotech.

1. Lipid-Based Delivery Vehicles

In some embodiments, nanoparticles may optionally comprise one or morelipids. In some embodiments, a nanoparticle may comprise a liposome. Insome embodiments, a nanoparticle may comprise a lipid bilayer. In someembodiments, a nanoparticle may comprise a lipid monolayer. In someembodiments, a nanoparticle may comprise a micelle. In some embodiments,a nanoparticle may comprise a core comprising a polymeric matrixsurrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer,etc.). In some embodiments, a nanoparticle may comprise a non-polymericcore (e.g., metal particle, quantum dot, ceramic particle, boneparticle, viral particle, etc.) surrounded by a lipid layer (e.g., lipidbilayer, lipid monolayer, etc.).

The percent of lipid in nanoparticles can range from 0% to 99% byweight, from 10% to 99% by weight, from 25% to 99% by weight, from 50%to 99% by weight, or from 75% to 99% by weight. In some embodiments, thepercent of lipid in nanoparticles can range from 0% to 75% by weight,from 0% to 50% by weight, from 0% to 25% by weight, or from 0% to 10% byweight. In some embodiments, the percent of lipid in nanoparticles canbe approximately 1% by weight, approximately 2% by weight, approximately3% by weight, approximately 4% by weight, approximately 5% by weight,approximately 10% by weight, approximately 15% by weight, approximately20% by weight, approximately 25% by weight, or approximately 30% byweight.

In some embodiments, lipids are oils. In general, any oil known in theart can be included in nanoparticles. In some embodiments, oil maycomprise one or more fatty acid groups or salts thereof. In someembodiments, a fatty acid group may comprise digestible, long chain(e.g., C8-050), substituted or unsubstituted hydrocarbons. In someembodiments, a fatty acid group may be a C10-C20 fatty acid or saltthereof. In some embodiments, a fatty acid group may be a C15-C20 fattyacid or salt thereof. In some embodiments, a fatty acid group may be aC15-C25 fatty acid or salt thereof. In some embodiments, a fatty acidgroup may be unsaturated. In some embodiments, a fatty acid group may bemonounsaturated. In some embodiments, a fatty acid group may bepolyunsaturated. In some embodiments, a double bond of an unsaturatedfatty acid group may be in the cis conformation. In some embodiments, adouble bond of an unsaturated fatty acid may be in the transconformation.

In some embodiments, a fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid. In some embodiments,the oil is a liquid triglyceride.

Suitable oils for use include plant oils and butyl stearate, caprylictriglyceride, capric triglyceride, cyclomethicone, diethyl sebacate,dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleylalcohol, silicone oil, and combinations thereof.

In some embodiments, a lipid is a hormone (e.g. estrogen, testosterone),steroid (e.g., cholesterol, bile acid), vitamin (e.g. vitamin E),phospholipid (e.g. phosphatidyl choline), sphingolipid (e.g. ceramides),or lipoprotein (e.g. apolipoprotein).

In certain embodiments, a lipid to be used in liposomes can be, but isnot limited to, one or a plurality of the following:phosphatidylcholine, lipid A, cholesterol, dolichol, sphingosine,sphingomyelin, ceramide, glycosylceramide, cerebroside, sulfatide,phytosphingosine, phosphatidyl-ethanolamine, phosphatidylglycerol,phosphatidylinositol, phosphatidylserine, cardiolipin, phosphatidicacid, and lyso-phophatides. In certain embodiments, a targeting moietycan be conjugated to the surface of a liposome.

In some embodiments, nanoparticle-stabilized liposomes are used todeliver the disclosed nucleic acid content. By allowing small chargednanoparticles (1 nm-30 nm) to adsorb on liposome surface,liposome-nanoparticle complexes have not only the merits of bareliposomes, but also tunable membrane rigidity and controllable liposomestability. When small charged nanoparticles approach the surface ofliposomes carrying either opposite charge or no net charge,electrostatic or charge-dipole interaction between nanoparticles andmembrane attracts the nanoparticles to stay on the membrane surface,being partially wrapped by lipid membrane. This induces local membranebending and globule surface tension of liposomes, both of which enabletuning of membrane rigidity. Moreover, adsorbed nanoparticles form acharged shell which protects liposomes against fusion, thereby enhancingliposome stability. In certain embodiments, small nanoparticles aremixed with liposomes under gentle vortex, and the nanoparticles stick toliposome surface spontaneously. In specific embodiments, smallnanoparticles can be, but are not limited to, polymeric nanoparticles,metallic nanoparticles, inorganic or organic nanoparticles, hybridsthereof, and/or combinations thereof.

In some embodiments, liposome-polymer nanoparticles are used to delivera combination of one or more inhibitory nucleic acids and one or morenucleic acids encoding a protein or polypeptide.

2. Lipid-Polymer Delivery Vehicles

In some embodiments, nanoparticles comprise one or more polymersassociated covalently, or non-covalently with one or more lipids. In thepreferred embodiments, nanoparticles comprise one or more phospholipids.

In some embodiments, a polymeric matrix can be surrounded by a coatinglayer (e.g., liposome, lipid monolayer, micelle, etc.). In oneembodiment, the lipid monolayer shell comprises an amphiphilic compound.In another embodiment, the amphiphilic compound is lecithin. In anotherembodiment, the lipid monolayer is stabilized.

Specific examples of amphiphilic compounds include, but are not limitedto, phospholipids, such as 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), diarachidoylphosphatidylcholine (DAPC),dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine(DTPC), and dilignoceroylphatidylcholine (DLPC), incorporated at a ratioof between 0.01-60 (weight lipid/w polymer), most preferably between0.1-30 (weight lipid/w polymer). Phospholipids which may be usedinclude, but are not limited to, phosphatidic acids, phosphatidylcholines with both saturated and unsaturated lipids, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines,phosphatidylinositols, lysophosphatidyl derivatives, cardiolipin, andβ-acyl-y-alkyl phospholipids. Examples of phospholipids include, but arenot limited to, phosphatidylcholines such asdioleoylphosphatidylcholine, dimyristoylphosphatidylcholine,dipentadecanoylphosphatidylcholine dilauroylphosphatidylcholine,dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), diarachidoylphosphatidylcholine (DAPC),dibehenoylphosphatidylcho-line (DBPC), ditricosanoylphosphatidylcholine(DTPC), dilignoceroylphatidylcholine (DLPC); andphosphatidylethanolamines such as dioleoylphosphatidylethanolamine or1-hexadecyl-2-palmitoylglycerophos-phoethanolamine. Syntheticphospholipids with asymmetric acyl chains (e.g., with one acyl chain of6 carbons and another acyl chain of 12 carbons) may also be used.

In a particular embodiment, an amphiphilic component that can be used toform an amphiphilic layer is lecithin, and, in particular,phosphatidylcholine. Lecithin is an amphiphilic lipid and, as such,forms a phospholipid bilayer having the hydrophilic (polar) heads facingtheir surroundings, which are oftentimes aqueous, and the hydrophobictails facing each other. Lecithin has an advantage of being a naturallipid that is available from, e.g., soybean, and already has FDAapproval for use in other delivery devices.

In certain embodiments, the amphiphilic layer of the nanoparticle, e.g.,the layer of lecithin, is a monolayer, meaning the layer is not aphospholipid bilayer, but exists as a single continuous or discontinuouslayer around, or within, the nanoparticle. A monolayer has the advantageof allowing the nanoparticles to be smaller in size, which makes themeasier to prepare. The amphiphilic layer is “associated with” thenanoparticle, meaning it is positioned in some proximity to thepolymeric matrix, such as surrounding the outside of the polymericmatrix (e.g., PLGA), or dispersed within the polymers that make up thenanoparticle.

By covering the polymeric nanoparticles with a thin film of smallmolecule amphiphilic compounds, the nanoparticles have merits of bothpolymer- and lipid-based nanoparticles, while excluding some of theirlimitations. The amphiphilic compounds form a tightly assembledmonolayer around the polymeric core. This monolayer effectively preventsthe carried agents from freely diffusing out of the nanoparticle,thereby enhancing the encapsulation yield and slowing drug release.Moreover, the amphiphilic monolayer also reduces water penetration rateinto the nanoparticle, which slows hydrolysis rate of the biodegradablepolymers, thereby increasing particle stability and lifetime.

In further embodiments, targeting ligands can be conjugated to theamphiphilic component prior to incorporating them into the nanoparticle,the composition of the nanoparticle and its surface properties can bemore accurately quantified. Alternatively, targeting ligands can beconjugated the polymeric component of the nanoparticles.

a. Lipid-Conjugated Polymers

In some embodiments, the nanoparticle comprises a polymeric matrix,wherein the polymeric matrix comprises a lipid-terminated polymer suchas polyalkylene glycol and/or a polyester. In some embodiments, thenanoparticle comprises an amphiphilic lipid-terminated polymer, where acationic and/or an amniotic lipid is conjugated to a hydrophobicpolymer. In one embodiment, the polymeric matrix compriseslipid-terminated PEG.

In some embodiments, the polymeric matrix comprises lipid-terminatedcopolymer. In another embodiment, the polymeric matrix compriseslipid-terminated PEG and PLGA.

In one embodiment, the lipid is 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof.In a preferred embodiment, the polymeric matrix comprisesDSPE-terminated PEG. The lipid-terminated PEG can then, for example, bemixed with PLGA to form a nanoparticle.

3. Amphiphilic Polymers

In some embodiments, long-circulating, optionally cell-penetrating, andstimuli-responsive nanopaticles for effective in vivo delivery oftherapeutic, prophylactic and/or diagnostic agents are used. In thepreferred embodiment, the NPs are made of an amphiphilic polymer, mostpreferably a PEGylated polymer, which shows a response to a stimulussuch as pH, temperature, or light, such as an ultra pH-responsivecharacteristic with a pKa close to the endosomal pH (6.0-6.5) (Wang Y etal, Nat Mater, 13, 204-212 (2014)). The polymer may include a targetingor cell penetrating or adhesion molecule such as a tumor-penetratingpeptide iRGD.

Stimuli responsive polymers are well known in the art. Stimuliresponsive amphiphilic polymers, especially those that can self-assemblyto form nanoparticles, are not. However, it is possible to make stimuliresponsive amphiphilic copolymers through selection of a hydrophilic orhydrophobic polymer component of the copolymer, or by modification ofthe hydrophilic or hydrophobic polymers.

The nanoparticles can be formed by self-assembly in an emulsion of anon-aqueous solvent with an aqueous solvent of a first amphiphilicpolymer containing a polymer represented by Formula I:

(X)_(m)—(Y)_(n)  Formula I

wherein, m and n are independently integers between one and 1000,inclusive. X is a hydrophobic polymer and Y is a hydrophilic polymer,and at least one of X, Y, or both, is stimuli-responsive. Optionally thenanoparticles are formed by self-assembly of a mixture of polymersrepresented by Formula I and a second polymer containing a polymerrepresented by Formula II:

(Q)_(c)-(R)_(d)  Formula II

wherein, c and d are independently integers between zero and 1000,inclusive, with the proviso that the sum (c+d) is greater than one. Qand R are independently hydrophilic or hydrophobic polymers. Optionally,the nanoparticles are formed by self-assembly of a mixture of polymersrepresented by Formula I and Formula II, wherein the polymer representedby Formula I, Formula II, or both, contains a ligand, wherein the ligandis a targeting ligand, an adhesion ligand, a cell-penetrating ligand, oran endosomal-penetrating ligand, with the proviso that the ligand isconjugated to the hydrophilic polymer.

In some embodiments, the nanoparticles are formed by self-assembly of amixture of first stimuli-responsive hydrophobic polymer and a secondpolymer containing a polymer represented by Formula III:

(S)_(e)-(T)_(f)  Formula III

wherein, e and f are independently integers between zero and 1000,inclusive, with the proviso that the sum (e+f) is greater than one. Sand T are independently a hydrophilic polymer or a hydrophobic polymer.Optionally, the first stimuli-response hydrophobic polymer, the polymerrepresented by Formula III, or both contains a ligand, wherein theligand is a targeting ligand, an adhesion ligand, a cell-penetratingligand, or an endosomal-penetrating ligand, with the proviso that theligand is conjugated to the hydrophilic polymer.

In some embodiments, the nanoparticles are formed by self-assembly of amixture of first stimuli-responsive hydrophilic polymer and a secondpolymer containing a polymer represented by Formula III:

(S)_(e)-(T)_(f)  Formula III

wherein, e and f are independently integers between zero and 1000,inclusive, with the proviso that the sum (e+f) is greater than one. Sand T are independently a hydrophilic polymer or a hydrophobic polymer.Optionally, the first stimuli-response hydrophilic polymer, the polymerrepresented by Formula III, or both contains a ligand, wherein theligand is a targeting ligand, an adhesion ligand, a cell-penetratingligand, or an endosomal-penetrating ligand, with the proviso that theligand is conjugated to the hydrophilic polymer.

Optionally, the polymers that form the nanoparticles contain linkersbetween the blocks of hydrophilic and hydrophobic polymers, between thehydrophilic polymer and ligand, or both.

Amphiphilic copolymers can spontaneously self-assemble in aqueoussolution to form NPs with hydrophobic inner core and hydrophilic outershells. The hydrophobic inner core can be used to deliver therapeuticand diagnostic agents including genes, proteins, chemotherapeutic drugs,or other small molecules. The incorporation of stimuli-responsivemoieties to the hydrophobic core can easily accomplish thespatiotemporal control over the macroscopic properties of NPs, andthereby the release of the encapsulated cargo at the desired site.

The amphiphilic polymers are responsive to a stimulus. This may be a pHchange, redox change, temperature change, exposure to light or otherstimuli, including binding to a target. The responsiveness may beimparted solely by the hydrophilic polymer, the hydrophobic polymer orthe conjugate per se. The nanoparticles are formed of a mixture or blendof polymers. Some may be the amphiphilic polymers, preferably copolymersof modified polyethylene glycol (PEG) and polyesters, such as variousforms of PLGA-PEG or PLA-PEG copolymers, collectively referred to hereinas “PEGylated polymers”, some hydrophobic polymer such as PLGA, PLA orPGA, and/or some may be hydrophilic polymer such as a PEG or PEGderivative. Some will be modified by conjugation to a targeting agent, acell adhesion or a cell penetrating peptide.

Besides amphiphilic copolymers, hydrophobic polymers can be also used todevelop stimuli-responsive NPs for various biomedical applications. Forthese hydrophobic polymers, their NPs are prepared by using the mixtureof the hydrophobic polymer and amphiphilic polymer or amphiphiliccompound. The amphiphilic compound can include, but is not limited to,one or a plurality of naturally derived lipids, lipid-like materials,surfactants, or synthesized amphiphilic compounds.

The length of hydrophilic and/or hydrophobic polymers can be optimizedto optimize encapsulation of agent to be delivered, i.e., encapsulationefficiency (EE %). As demonstrated in the examples, as the PDPA lengthincreases, the EE % and size of the resulting NPs increase (Table 3),possibly because the increased PDPA length leads to an increase in thesize of the hydrophobic core. Specifically, the EE % reaches almost 100%for the polymer with 80 (PDPA80) or 100 (PDPA100) DPA repeat units.Notably, using a mixture of Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) (90 mol %)and tumor-penetrating polymer (iRGD-PEG-b-PDPA, 10 mol %) to prepare NPsdoes not cause obvious change in the EE % or particle size.

The amphiphilic polymers include a hydrophilic polymer. This ispreferably at an end which can orient to the exterior of thenanoparticles when formed by emulsion techniques such as self-assembly.

Polymers and copolymers that can be used to make the nanoparticlesdisclosed herein include, but are not limited to, polymers includingglycolic acid units, referred to herein as “PGA”, and lactic acid units,such as poly-L-Iactic acid, poly-D-Iactic acid, poly-D,L-Iactic acid,poly-L-Iactide, poly-D-Iactide, and poly-D,L-Iactide, collectivelyreferred to herein as “PLA”, and caprolactone units, such aspoly(8-caprolactone), collectively referred to herein as “PCL”; andcopolymers including lactic acid and glycolic acid units, such asvarious forms of poly(lactic acid-co-glycolic acid) andpoly(lactide-co-glycolide) characterized by the ratio of lacticacid:glycolic acid, collectively referred to herein as “PLGA”;polyacrylates, polyanhydrides, poly (ester anhydrides),poly-4-hydroxybutyrate (P4HB) combinations and derivatives thereof.

The polymer is preferably a biocompatible polymer. One simple test todetermine biocompatibility is to expose a polymer to cells in vitro;biocompatible polymers are polymers that typically will not result insignificant cell death at moderate concentrations, e.g., atconcentrations of 50 micrograms/10⁶ cells. For instance, a biocompatiblepolymer may cause less than about 20% cell death when exposed to cellssuch as fibroblasts or epithelial cells, even if phagocytosed orotherwise uptaken by such cells.

The biocompatible polymer is preferably biodegradable, i.e., the polymeris able to degrade, chemically and/or biologically, within aphysiological environment, such as within the body.

In some embodiments, the delivery vehicles comprise amphiphile-polymerparticles, e.g., comprising a water-insoluble polymeric core and apayload and at least one amphiphile within the core, as described inWO2016/065306, which is incorporated herein by reference in itsentirety.

In preferred embodiments, the nanoparticles comprise a core of mRNAcomplexed with cationic lipid-like compound G0-C14 andpoly(lactic-co-glycolic acid) (PLGA) polymer, coated with alipid-poly(ethylene glycol) (lipid-PEG) shell, e.g., (e.g., DSPE-PEG(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy{polyethylene glycol}]) or ceramide-PEG(N-palmitoyl-sphingosine-1-(succinyl{methoxy[polyethylene glycol]}) withPEG molecular weight (MW) 2000-5000)⁴⁵ (see, e.g., FIG. 1A herein).G0-C14 can be used for mRNA complexation, and PLGA, a widely clinicallyused biodegradable and biocompatible polymer, provides a stable NP core.

a. Stimuli that the Polymers can be Responsive to

The polymers can be responsive to changes in pH-, redox-, light-,temperature-, enzyme-, ultrasound, or other stimuli such as aconformation change resulting from binding.

Almeida, et al. J. Applied Pharm.l Sci. 02 (06)01-10 (2012) is anexcellent review of stimuli responsive polymers. The signs or stimulithat trigger the structural changes on smart polymers can be classifiedin three main groups: physical stimuli (temperature, ultrasound, light,mechanical stress), chemical stimuli (pH and ionic strength) andbiological stimuli (enzymes and bio molecules).

Stimuli can be artificially controlled (with a magnetic or electricfield, light, ultrasounds, etc.) or naturally promoted by internalphysiological environment through a feedback mechanism, leading tochanges in the polymer net that allow the drug delivery without anyexternal intervention (for example: pH changes in certain vital organsor related to a disease; temperature change or presence of enzymes orother antigens) or by the physiological condition. In the presence of asign or stimuli, changes can happen on the surface and solubility of thepolymer as well as on sol-gel transition.

Smart polymers can be classified according to the stimuli they respondto or to their physical features. Regarding the physical shape, they canbe classified as free linear polymer chain solutions, reversible gelscovalently cross-linked and polymer chain grafted to the surface.

Stimuli responsive polymers are also reviewed by James, et al., ActaPharma. Sinica B 4(2):120-127 (2014). The following is a list ofexemplary polymers categorized by responsive to various stimuli:

-   -   Temperature: POLOXAMERS, poly(N-alkylacrylamide)s,        poly(N-vinylcaprolactam)s, cellulose, xyoglucan, and chitosan    -   pH: poly(methacrylic acid)s, poly(vinylpyridine)s, and        poly(vinylimmidazole)s    -   light: modified poly(acrylamide)s    -   electric field: sulfonated polystyrenes, poly(thiophene)s, and        poly(ethyloxazoline)s    -   ultrasound: ethylenevinylacetate

These transitions are reversible and include changes in physical state,shape and solubility, solvent interactions, hydrophilic and lipophilicbalances and conductivity. The driving forces behind these transitionsinclude neutralization of charged groups by the addition of oppositelycharged polymers or by pH shift, and change in thehydrophilic/lipophilic balance or changes in hydrogen bonding due toincrease or decrease in temperature. Responses of a stimulus-responsivepolymer can be of various types. Responsiveness of a polymeric solutioninitiated by physical or chemical stimuli is limited to the destructionand formation of various secondary forces including hydrogen bonding,hydrophobic forces, van der Waals forces and electrostatic interaction.Chemical events include simple reactions such as oxidation, acid-basereaction, reduction and hydrolysis of moieties attached to the polymerchain. In some cases, dramatic conformational change in the polymericstructure occurs, e.g., degradation of the polymeric structure due toirreversible bond breakage in response to an external stimulus.

b. pH Dependent Polymers

Exemplary pH dependent polymers include dendrimers formed ofpoly(lysine), poly(hydroxyproline), PEG-PLA, Poly(propyl acrylic acid),Poly(ethacrylic acid), CARBOPOLL®, Polysilamine, EUDRAGIT® S-100EUDRAGIT® L-100, Chitosan, PMAA-PEG copolymer, sodium alginate (Ca2+).The ionic pH sensitive polymers are able to accept or release protons inresponse to pH changes. These polymers contain acid groups (carboxylicor sulfonic) or basic groups (ammonium salts) so that the pH sensitivepolymers are polyelectrolytes that have in their structure acid or basicgroups that can accept or release protons in response to pH changes inthe surrounding environment. pH values from several tissues and cellcompartments can be used to trigger release in these tissues. Forexample, the pH of blood is 7.4-7.5; stomach is 1.0-3.0; duodenum is4.8-8.2; colon is 7.0-7.5; lysosome is 4.5-5.0; Golgi complex is 6.4;tumor—extracellular medium is 6.2-7.2.

Examples of these polymers include poly(acrylic acid) (PAA) (CARBOPOLI®)and derivatives, poly(methacrylic acid) (PMAA), poly(2-(diisopropylamino) ethylmethacrylate) (PDPA), poly(2-(hexamethyleneimino) ethyl methacrylate), poly(2-diethylaminoethylmethacrylate) (PDEAEMA), poly(ethylene imine), poly(L-lysine) andpoly(N,N-dimethylaminoethylmetha crylate) (PDMAEMA). Polymers withfunctional acid groups pH sensitive polymers include poly(acrylic acid)(PAA) or poly(methacrylic) acid (PMAA) are polyanions that have in theirstructure a great number of ionizable acid groups, like carboxylic acidor sulfonic acid. The pH in which acids become ionized depends on thepolymer's pKa (depends on the polymer's composition and molecularweight). Polymers with functional basic groups include polycations suchas poly(4-vinylpyridine), poly(2-vinylpyridine) (PVP) andpoly(vinylamine) (PVAm), are protonated at high pH values and positivelyionized at neutral or low pH values, i.e., they go through a phasetransition at pH 5 due to the deprotonation of the pyridine groups.Other polybases are poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA)and poly(2-diethylaminoethyl methacrylate) (PDEAEMA), with amino groupsin their structure which in acid environments gain protons, and in basicenvironments release the protons. Examples of polycationicpolyelectrolyte polymers are poly(N,N-diakyl aminoethyl methacrylate),poly(lysine) (PL), poly(ethylenimine) (PEI) and chitosan. Commerciallyavailable polymers include EUDRAGIT L® and EUDRAGIT S® from Röhm PharmaGmBH (with methacrylic acid and methylmethacrylate in theircomposition), CMEC (a cellulose derivative) from Freund Sangyo Co., CAPby Wako Pure Chemicals Ltd., HP-50 and ASM by Shin-Etsu Chemical Co.,Ltd.

There are several natural polymers (for example, albumin, gelatin andchitosan) that present pH sensibility. Chitosan is a cationic aminopolysaccharide, derivative from chitin, which is biocompatible andresorbable. Additional examples include the anionic polymer PEAA(polyethacrylic acid) or by PPAA (polypropyl acrylic acid),Polypropylacrylic acid (PPAA) and polyethacrylic acid (PEAA), andpoly(ethylene glycol)-poly(aspartame hydrazine doxorubicin)[(PEG-p(Asp-Hid-dox), and polycationic polymers, such aspoly(2-diethylaminoethyl methacrylate) (PDEAEMA).

c. Temperature Dependent Polymers

Temperature dependent polymers are sensitive to the temperature andchange their microstructural features in response to change intemperature. Thermo-responsive polymers present in their structure avery sensitive balance between the hydrophobic and the hydrophilicgroups and a small change in the temperature can create new adjustments.If the polymeric solution has a phase below the critical solutiontemperature, it will become insoluble after heating. Above the criticalsolution temperature (LCST), the interaction strengths (hydrogenlinkages) between the water molecules and the polymer becomeunfavorable, it dehydrates and a predominance of the hydrophobicinteraction occurs, causing the polymer to swell. The LSCT is thecritical temperature in which the polymeric solution shows a phaseseparation, going from one phase (isotropic state) to two phases(anisotropic state). The accumulation of temperature sensitive polymericsystems in solid tumors is due to the increased impermeability effect tothe tumor vascular net retention and to the use of an external impulse(heat source) on the tumor area. This temperature increase promotes thechanging of the microstructure of the polymeric system, turning it intogel and releasing the drug, thus increasing the drug in theintra-tumoral area and the therapeutic efficiency, and reducing the sideeffects (MacEwan et al., 2010).

Examples of thermosensitive polymers include the poly(N-substitutedacrylamide) polymers such as poly(N-isopoprylacrilamide) (PNIPAAm), poly(N,N′-diethyl acrylamide), poly (dimethylamino ethyl methacrylate andpoly (N-(L)-(1-hydroxymethyl) propyl methacrylamide). Other examples ofthermo-responsive polymers are: copolymers blocks of poly(ethyleneglycol)/poly(lactide-coglicolide) (PEG/PLGA, REGEL®),polyoxyethylenepolyoxypropylene (PEO/PPO), triple blocks of copolymerspolyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPOPEO) andpoly(ethylene glycol)-poly(lactic acid)-poly(ethylene glycol)(PEG-PLA-PEG). Exemplary polymers and their LCST: PNIPAAm, LCST 32° C.;PDEAAm, LCST 26-35° C.; PDMAEMA, LCST 50° C.;poly(N-(L)-(hydroxymethyl)propylmethacrylamide), LCST 30° C.

An increase of the hydrophobic monomers (as, for example, the butylmethacrylate) or on the molecular weight, results in a LCST decrease(Jeong, Gutowska, 2002). The incorporation of hydrophilic monomers suchas acrylic acid or hydroxyethyl methacrylate) fosters the creation ofincreases LCST. The co-polymers NIPAAm conjugated with hydrophilicunities such as acrylic acid promotes the increase of LCST totemperatures around 37° C., i.e., the body temperature. Polymers with2-hydroxyethyl (methacrylate) (HEMA) promote the increase of LCST abovethe body temperature

POLOXAMERs and derivatives are well known temperature sensitivepolymers. The copolymer blocks based on PEO-PPO sequences constitutesone family of triple blocks of commercialized copolymers with thefollowing names: PLURONICS®, POLOXAMERS® AND TETRONICS®. POLOXAMERS® arenon-ionic polymers polyoxyethylenepolyoxypropylene-polyoxyethylene(PEOn-PPOn-PEOn), with many pharmaceutical uses (Ricci et al., 2005).The triple block of copolymers PEO—PPO-PEO (PLURONICS® or POLOXAMERS®)get into gel at body temperature in concentrations above 15% (m/m). ThePOLOXAMERs® normally used are: 188 (F-68), 237 (F-87), 338 (F-108) and407 (F-127). “F” refers to the polymer in the form of flakes. PLURONICS®and TETRONICS® are polymers approved by FDA to be used as foodadditives, pharmaceutical ingredients, drug carriers in parenteralsystems, tissue engineering and agricultural products. PLURONIC F-127(Polaxamer 407, PF-127) can also be used as carrier in several routes ofadministration, including oral, cutaneous, intranasal, vaginal, rectal,ocular and parenteral. PLURONIC® F127 (PF-127) or POLOXAMER 407 (P407)(copolymer polyoxyethylene 106-polyoxypropylene 70-polyoxyethylene106)contains about 70% of ethylene oxide which contributes to itshydrophilicity.

d. Polymers with Dual Stimuli-Responsiveness

To obtain a temperature and pH sensitive polymer it is only necessary tocombine temperature sensitive monomers (as, for example,poly(N-isopropylacrylamide-co-methacrylic acid and PNIPAm) with pHsensitive monomers (as, for example, AA and MAA).

e. Polymers with Binding or Biological Responsiveness

Biologically responsive polymer systems are increasingly important invarious biomedical applications. The major advantage of bioresponsivepolymers is that they can respond to the stimuli that are inherentlypresent in the natural system. Bioresponsive polymeric systems mainlyarise from common functional groups that are known to interact withbiologically relevant species, and in other instances the syntheticpolymer is conjugated to a biological component. Bioresponsive polymersare classified into antigen-responsive polymers, glucose-sensitivepolymers, and enzyme-responsive polymers.

Glucose-responsive polymeric-based systems have been developed based onthe following approaches: enzymatic oxidation of glucose by glucoseoxidase, and binding of glucose with lectin or reversible covalent bondformation with phenylboronic acid moieties. Glucose sensitivity occursby the response of the polymer toward the byproducts that result fromthe enzymatic oxidation of glucose. Glucose oxidase oxidises glucoseresulting in the formation of gluconic acid and H₂O₂. For example, inthe case of poly (acrylicacid) conjugated with the GOx system, as theblood glucose level is increased glucose is converted into gluconic acidwhich causes the reduction of pH and protonation of PAA carboxylatemoieties, facilitating the release of insulin. Another system utilizesthe unique carbohydrate binding properties of lectin for the fabricationof a glucose-sensitive system. Concanavalin A (Con A) is a lectinpossessing four binding sites and has been used frequently ininsulin-modulated drug delivery. In this type of system the insulinmoiety is chemically modified by introducing a functional group (orglucose molecule) and then attached to a carrier or support throughspecific interactions which can only be interrupted by the glucoseitself. The glycosylated insulin-Con A complex exploits the competitivebinding behaviour of Con A with glucose and glycosylated insulin. Thefree glucose molecule causes the displacement of glycosylated ConA-insulin conjugates.

Another approach includes polymers with phenylboronic groups and polyolpolymers that form a gel through complex formation between the pendantphenylborate and hydroxyl groups. Instead of polyol polymers, shortmolecules such as diglucosylhexadiamine have been used. As the glucoseconcentration increases, the crosslinking density of the gel decreasesand as a result insulin is released from the eroded gel. The glucoseexchange reaction is reversible and reformation of the gel occurs as aresult of borate-polyol crosslinking.

Field-responsive polymers respond to the application of electric,magnetic, sonic or electromagnetic fields. The additional benefit overtraditional stimuli-sensitive polymers is their fast response time,anisotropic deformation due to directional stimuli, and also acontrolled drug release rate simply by modulating the point of signalcontrol.

f. Light-Sensitive Polymers

A light-sensitive polymer undergoes a phase transition in response toexposure to light. These polymers can be classified into UV-sensitiveand visible-sensitive systems on the basis of the wavelength of lightthat triggers the phase transition.

A variety of materials are known, such as a leuco-derivative molecule,bis(4-dimethylamino)phenylmethyl leucocyanide, which undergoes phasetransition behaviour in response to UV light. Triphenylmethane-leucoderivatives dissociate into ion-pairs such as triphenylmethyl cationsupon UV irradiation. At a fixed temperature these hydrogels swelldiscontinuously due to increased osmotic pressure in response to UVirradiation but shrink when the stimulus is removed. Another example isa thermosensitive diarylated pluronic F-127.

Visible light-sensitive polymeric materials can be prepared byincorporating photosensitive molecules such as chromophores (e.g.,trisodium salt of copper chlorophyllin). When light of appropriatewavelength is applied, the chromophore absorbs light which is thendissipated locally as heat by radiationless transition, increasing thelocal temperature of the polymeric material, leading to alteration ofthe swelling behavior. The temperature increase directly depends on thechromophore concentration and light intensity.

g. Electric Field-Sensitive Polymers

Electric field-sensitive polymers change their physical properties inresponse to a small change in electric current. These polymers contain arelatively large concentration of ionisable groups along the back bonechain that are also pH-responsive. Electro-responsive polymers transformelectric energy into mechanical energy. The electric current causes achange in pH which leads to disruption of hydrogen bonding betweenpolymer chains, causing degradation or bending of the polymer chain.Major mechanisms involved in drug release from electro-responsivepolymer are diffusion, electrophoresis of charged drug, forcedconvection of drug out of the polymer or degradation of the polymer.

Naturally occurring polymers such as chitosan, alginate and hyalouronicacid are commonly employed to prepare electro-responsive materials.Major synthetic polymers that have been used include allyl amine, vinylalcohol, acrylonitrile, methacrylic acid and vinylacrylic acid. In somecases, combinations of natural and synthetic polymers have been used.Most polymers that exhibit electro-sensitive behavior arepolyelectrolytes and undergo deformation under an electric field due toanisotropic swelling or deswelling as the charged ions move towards thecathode or anode. Neutral polymers that exhibit electro-sensitivebehavior require the presence of a polarisable component with theability to respond to the electric field. Another example of a materialwhich can be used is poly(2-acrylamido-2-methylpropane sulphonicacid-co-n-butylmethacrylate).

4. Hydrogel-Forming Polymers

In some embodiments, the delivery vehicles for the nucleic acids areformed from a biocompatible, hydrogel-forming polymer encapsulating thenucleic acids to be delivered. In some embodiments, the hydrogel is ananionic polymer that is cross-linked with a polycationic polymer. Insome embodiments the nanoparticles are conFIG.d with a core and envelopestructure. In these embodiments, the nucleic acids are preferablyencapsulated in the core hydrogel and the drug-loaded polymericparticles are encapsulated within the envelope hydrogel. In preferredembodiments, the core and envelope hydrogels are separated by a membraneor shell.

Examples of materials which can be used to form a suitable hydrogelinclude polysaccharides such as alginate, polyphosphazines, poly(acrylicacids), poly(methacrylic acids), poly(alkylene oxides), poly(vinylacetate), polyvinylpyrrolidone (PVP), and copolymers and blends of each.See, for example, U.S. Pat. Nos. 5,709,854, 6,129,761 and 6,858,229.

In general, these polymers are at least partially soluble in aqueoussolutions, such as water, buffered salt solutions, or aqueous alcoholsolutions, that have charged side groups, or a monovalent ionic saltthereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), poly(vinyl acetate), and sulfonated polymers,such as sulfonated polystyrene. Copolymers having acidic side groupsformed by reaction of acrylic or methacrylic acid and vinyl ethermonomers or polymers can also be used. Examples of acidic groups arecarboxylic acid groups and sulfonic acid groups.

Examples of polymers with basic side groups that can be reacted withanions are poly(vinyl amines), poly(vinyl pyridine), poly(vinylimidazole), and some imino substituted polyphosphazenes. The ammonium orquaternary salt of the polymers can also be formed from the backbonenitrogens or pendant imino groups. Examples of basic side groups areamino and imino groups.

The biocompatible, hydrogel-forming polymer is preferably awater-soluble gelling agent. In preferred embodiments, the water-solublegelling agent is a polysaccharide gum, more preferably a polyanionicpolymer.

In some embodiments, the targeting ligands are covalently attached tohydrogel-forming polymers. In some embodiments, the nucleic acids to betargeted are attached to the hydrogel forming polymer via a linkingmoiety that is designed to be cleaved in vivo. The composition of thelinking moiety can also be selected in view of the desired release rateof the nucleic acids.

5. Moieties Attached to Particles

The nanoparticles or other delivery vehicles can include bindingmoieties or targeting moieties that specifically bind to a target cellor tissue. Representative targeting moieties include, but are notlimited to, antibodies and antigen binding fragments thereof, aptamers,peptides, and small molecules. The binding moiety can be conjugated to apolymer that forms the nanoparticle. Typically the binding moiety isdisplayed on the outer shell of the nanoparticle. The outer shell servesas a shield to prevent the nanoparticles from being recognized by asubject's immune system thereby increasing the half-life of thenanoparticles in the subject. The nanoparticles also contain ahydrophobic core. In preferred embodiments, the hydrophobic core is madeof a biodegradable polymeric material. The inner core carriestherapeutic payloads and releases the therapeutic payloads at asustained rate after systemic, intraperitoneal, oral, pulmonary, ortopical administration. The nanoparticles also optionally include adetectable label, for example a fluorophore or NMR contrast agent thatallows visualization of nanoparticles within plaques.

The targeting moiety of the nanoparticle can be an antibody or antigenbinding fragment thereof. The targeting moieties should have an affinityfor a cell-surface receptor or cell-surface antigen on the target cells.The targeting moieties may result in internalization of the particlewithin the target cell.

The targeting moiety can specifically recognize and bind to a targetmolecule specific for a cell type, a tissue type, or an organ. Thetarget molecule can be a cell surface polypeptide, lipid, or glycolipid.The target molecule can be a receptor that is selectively expressed on aspecific cell surface, a tissue or an organ. Cell specific markers canbe for specific types of cells including, but not limited to stem cells,skin cells, blood cells, immune cells, muscle cells, nerve cells, cancercells, virally infected cells, and organ specific cells. The cellmarkers can be specific for endothelial, ectodermal, or mesenchymalcells. Representative cell specific markers include, but are not limitedto cancer specific markers.

Exemplary targets include PSMA; GAH; HER2; Tf receptor; EpCAM; gC1qR(p32); Nucleolin; avβ3/5; Collagen IV; Fibronectin; FA receptor; andMitochondria. Exemplary methods and moieties for targeting cancer cells,including proteins, peptides, nucleic acid-based ligands and smallmolecules, are described below and in Bertrand et al., Adv Drug DelivRev. 2014 February; 66: 2-25 (see esp. table 2 and section 3.4,“Targeting Ligands”).

a. Peptide Targeting Moieties

In a preferred embodiment, the targeting moiety is a peptide.Specifically, the plaque targeted peptide can be, but is not limited to,one or more of the following: RGD, iRGD(CRGDK/RGPD/EC), LyP-1,P3(CKGGRAKDC), or their combinations at various molar ratios. Thetargeting peptides can be covalently associated with the polymer and thecovalent association can be mediated by a linker. The peptides target toactively growing (angiogenic) vascular endothelial cells. Thoseangiogenic endothelial cells frequently appear in metabolic tissues suchas adipose tissues.

b. Antibody Targeting Moieties

The targeting moiety can be an antibody or an antigen-binding fragmentthereof. The antibody can be any type of immunoglobulin that is known inthe art. For instance, the antibody can be of any isotype, e.g., IgA,IgD, IgE, IgG, IgM, etc. The antibody can be monoclonal or polyclonal.The antibody can be a naturally-occurring antibody, e.g., an antibodyisolated and/or purified from a mammal, e.g., mouse, rabbit, goat,horse, chicken, hamster, human, etc. Alternatively, the antibody can bea genetically-engineered antibody, e.g., a humanized antibody or achimeric antibody. The antibody can be in monomeric or polymeric form.The antigen binding portion of the antibody can be any portion that hasat least one antigen binding site, such as Fab, F(ab′)₂, dsFv, sFv,diabodies, and triabodies. In certain embodiments, the antibody is asingle chain antibody.

c. Aptamer Targeting Moieties

Aptamers are oligonucleotide or peptide sequences with the capacity torecognize virtually any class of target molecules with high affinity andspecificity. Aptamers bind to targets such as small organics, peptides,proteins, cells, and tissues. Unlike antibodies, some aptamers exhibitstereoselectivity. The aptamers can be designed to bind to specifictargets expressed on cells, tissues or organs.

d. Additional Moieties

The nanoparticles can contain one or more polymer conjugates containingend-to-end linkages between the polymer and a moiety. The moiety can bea targeting moiety, a detectable label, or a therapeutic, prophylactic,or diagnostic agent. For example, a polymer conjugate can be aPLGA-PEG-phosphonate. The additional targeting elements may refer toelements that bind to or otherwise localize the nanoparticles to aspecific locale. The locale may be a tissue, a particular cell type, ora subcellular compartment. The targeting element of the nanoparticle canbe an antibody or antigen binding fragment thereof, an aptamer, or asmall molecule (less than 500 Daltons). The additional targetingelements may have an affinity for a cell-surface receptor orcell-surface antigen on a target cell and result in internalization ofthe particle within the target cell.

Pharmaceutical Compositions and Methods of Administration

The methods described herein include the use of pharmaceuticalcompositions comprising an mRNA encoding a tumor suppressor complexedwith a delivery vehicle as an active ingredient.

Pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes saline, solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions, e.g., an immunotherapy agent as described herein.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration.

Methods of formulating suitable pharmaceutical compositions are known inthe art, see, e.g., Remington: The Science and Practice of Pharmacy,21st ed., 2005; and the books in the series Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs (Dekker, N.Y.). Forexample, solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays or suppositories. For transdermal administration, the activecompounds are formulated into ointments, salves, gels, or creams asgenerally known in the art.

The pharmaceutical compositions can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Immunotherapy

In some embodiments, the methods also include co-administering animmunotherapy agent to a subject who is treated with a method orcomposition described herein. Immunotherapy agents include thosetherapies that target tumor-induced immune suppression; see, e.g.,Stewart and Smyth, Cancer Metastasis Rev. 2011 March; 30(1):125-40.

Examples of immunotherapies include, but are not limited to, adoptive Tcell therapies or cancer vaccine preparations designed to induce Tlymphocytes to recognize cancer cells, as well as checkpoint inhibitorssuch as anti-CD137 (BMS-663513), anti-PD1 (e.g., Nivolumab,pembrolizumab/MK-3475, Pidilizumab (CT-011)), anti-PDL1 (e.g.,BMS-936559, MPDL3280A), or anti-CTLA-4 (e.g., ipilumimab; see, e.g.,Kruger et al., Histol Histopathol. 2007 June; 22(6):687-96; Eggermont etal., Semin Oncol. 2010 October; 37(5):455-9; Klinke D J., Mol Cancer.2010 Sep. 15; 9:242; Alexandrescu et al., J Immunother. 2010July-August; 33(6):570-90; Moschella et al., Ann N Y Acad Sci. 2010April; 1194:169-78; Ganesan and Bakhshi, Natl Med J India. 2010January-February; 23(1):21-7; Golovina and Vonderheide, Cancer J. 2010July-August; 16(4):342-7.

Exemplary anti-PD-1 antibodies that can be used in the methods describedherein include those that bind to human PD-1; an exemplary PD-1 proteinsequence is provided at NCBI Accession No. NP_005009.2. Exemplaryantibodies are described in U.S. Pat. Nos. 8,008,449; 9,073,994; andUS20110271358, including PF-06801591, AMP-224, BGB-A317, BI 754091,JS001, MEDI0680, PDR001, REGN2810, SHR-1210, TSR-042, pembrolizumab,nivolumab, avelumab, pidilizumab, and atezolizumab.

Exemplary anti-CD40 antibodies that can be used in the methods describedherein include those that bind to human CD40; exemplary CD40 proteinprecursor sequences are provided at NCBI Accession No. NP_001241.1,NP_690593.1, NP_001309351.1, NP_001309350.1 and NP_001289682.1.Exemplary antibodies include those described in WO2002/088186;WO2007/124299; WO2011/123489; WO2012/149356; WO2012/111762;WO2014/070934; US20130011405; US20070148163; US20040120948;US20030165499; U.S. Pat. No. 8,591,900; including dacetuzumab,lucatumumab, bleselumab, teneliximab, ADC-1013, CP-870,893, Chi Lob 7/4,HCD122, SGN-4, SEA-CD40, BMS-986004, and APX005M. In some embodiments,the anti-CD40 antibody is a CD40 agonist, and not a CD40 antagonist.

Exemplary anti-PD-L1 antibodies that can be used in the methodsdescribed herein include those that bind to human PD-L1; exemplary PD-L1protein sequences are provided at NCBI Accession No. NP_001254635.1,NP_001300958.1, and NP_054862.1. Exemplary antibodies are described inUS20170058033; WO2016/061142A1; WO2016/007235A1; WO2014/195852A1; andWO2013/079174A1, including BMS-936559 (MDX-1105), FAZ053, KN035,Atezolizumab (Tecentriq, MPDL3280A), Avelumab (Bavencio), and Durvalumab(Imfinzi, MEDI-4736).

In some embodiments, these immunotherapies may primarily targetimmunoregulatory cell types such as regulatory T cells (Tregs) or M2polarized macrophages, e.g., by reducing number, altering function, orpreventing tumor localization of the immunoregulatory cell types. Forexample, Treg-targeted therapy includes anti-GITR monoclonal antibody(TRX518), cyclophosphamide (e.g., metronomic doses), arsenic trioxide,paclitaxel, sunitinib, oxaliplatin, PLX4720, anthracycline-basedchemotherapy, Daclizumab (anti-CD25); Immunotoxin eg. Ontak (denileukindiftitox); lymphoablation (e.g., chemical or radiation lymphoablation)and agents that selectively target the VEGF-VEGFR signaling axis, suchas VEGF blocking antibodies (e.g., bevacizumab), or inhibitors of VEGFRtyrosine kinase activity (e.g., lenvatinib) or ATP hydrolysis (e.g.,using ectonucleotidase inhibitors, e.g., ARL67156(6-N,N-Diethyl-D-β,γ-dibromomethyleneATP trisodium salt),8-(4-chlorophenylthio) cAMP (pCPT-cAMP) and a related cyclic nucleotideanalog (8-[4-chlorophenylthio] cGMP; pCPT-cGMP) and those described inWO 2007135195, as well as mAbs against CD73 or CD39). Docetaxel also haseffects on M2 macrophages. See, e.g., Zitvogel et al., Immunity 39:74-88(2013).

In another example, M2 macrophage targeted therapy includesclodronate-liposomes (Zeisberger, et al., Br J Cancer. 95:272-281(2006)), DNA based vaccines (Luo, et al., J Clin Invest. 116(8):2132-2141 (2006)), and M2 macrophage targeted pro-apoptotic peptides(Cieslewicz, et al., PNAS. 110(40): 15919-15924 (2013)). Some usefulimmunotherapies target the metabolic processes of immunity, and includeadenosine receptor antagonists and small molecule inhibitors, e.g.,istradefylline (KW-6002) and SCH-58261; indoleamine 2,3-dioxygenase(IDO) inhibitors, e.g., Small molecule inhibitors (e.g.,1-methyl-tryptophan (IMT), 1-methyl-d-tryptophan (D1MT), and Toho-1) orIDO-specific siRNAs, or natural products (e.g., Brassinin or exiguamine)(see, e.g., Munn, Front Biosci (Elite Ed). 2012 Jan. 1; 4: 734-45) ormonoclonal antibodies that neutralize the metabolites of IDO, e.g., mAbsagainst N-formyl-kynurenine.

In some embodiments, the immunotherapies may antagonize the action ofcytokines and chemokines such as IL-10, TGF-beta, IL-6, CCL2 and othersthat are associated with immunosuppression in cancer. For example,TGF-beta neutralizing therapies include anti-TGF-beta antibodies (e.g.fresolimumab, Infliximab, Lerdelimumab, GC-1008), antisenseoligodeoxynucleotides (e.g., Trabedersen), and small molecule inhibitorsof TGF-beta (e.g. LY2157299), (Wojtowicz-Praga, Invest New Drugs. 21(1):21-32 (2003)). Another example of therapies that antagonizeimmunosuppression cytokines can include anti-IL-6 antibodies (e.g.siltuximab) (Guo, et al., Cancer Treat Rev. 38(7):904-910 (2012). mAbsagainst IL-10 or its receptor can also be used, e.g., humanized versionsof those described in Llorente et al., Arthritis & Rheumatism, 43(8):1790-1800, 2000 (anti-IL-10 mAb), or Newton et al., Clin Exp Immunol.2014 July; 177(1):261-8 (Anti-interleukin-10R1 monoclonal antibody).mAbs against CCL2 or its receptors can also be used. In someembodiments, the cytokine immunotherapy is combined with a commonly usedchemotherapeutic agent (e.g., gemcitabine, docetaxel, cisplatin,tamoxifen) as described in U.S. Pat. No. 8,476,246.

In some embodiments, immunotherapies can include agents that arebelieved to elicit “danger” signals, e.g., “PAMPs” (pathogen-associatedmolecular patterns) or “DAMPs” (damage-associated molecular patterns)that stimulate an immune response against the cancer. See, e.g., Pradeuand Cooper, Front Immunol. 2012, 3:287; Escamilla-Tilch et al., ImmunolCell Biol. 2013 November-December; 91(10):601-10. In some embodiments,immunotherapies can agonize toll-like receptors (TLRs) to stimulate animmune response. For example, TLR agonists include vaccine adjuvants(e.g., 3M-052) and small molecules (e.g., Imiquimod, muramyl dipeptide,CpG, and mifamurtide (muramyl tripeptide)) as well as polysaccharidekrestin and endotoxin. See, Galluzi et al., Oncoimmunol. 1(5): 699-716(2012), Lu et al., Clin Cancer Res. Jan. 1, 2011; 17(1): 67-76, U.S.Pat. Nos. 8,795,678 and 8,790,655. In some embodiments, immunotherapiescan involve administration of cytokines that elicit an anti-cancerimmune response, see Lee & Margolin, Cancers. 3: 3856-3893(2011). Forexample, the cytokine IL-12 can be administered (Portielje, et al.,Cancer Immunol Immunother. 52: 133-144 (2003)) or as gene therapy(Melero, et al., Trends Immunol. 22(3): 113-115 (2001)). In anotherexample, interferons (IFNs), e.g., IFNgamma, can be administered asadjuvant therapy (Dunn et al., Nat Rev Immunol. 6: 836-848 (2006)).

In some embodiments, immunotherapies can antagonize cell surfacereceptors to enhance the anti-cancer immune response. For example,antagonistic monoclonal antibodies that boost the anti-cancer immuneresponse can include antibodies that target CTLA-4 (ipilimumab, seeTarhini and Iqbal, Onco Targets Ther. 3:15-25 (2010) and U.S. Pat. No.7,741,345 or Tremelimumab) or antibodies that target PD-1 (nivolumab,see Topalian, et al., NEJM. 366(26): 2443-2454 (2012) andWO2013/173223A1, pembrolizumab/MK-3475, Pidilizumab (CT-011)).

Some immunotherapies enhance T cell recruitment to the tumor site (suchas Endothelin receptor-A/B (ETRA/B) blockade, e.g., with macitentan orthe combination of the ETRA and ETRB antagonists BQ123 and BQ788, seeCoffman et al., Cancer Biol Ther. 2013 February; 14(2):184-92), orenhance CD8 T-cell memory cell formation (e.g., using rapamycin andmetformin, see, e.g., Pearce et al., Nature. 2009 Jul. 2;460(7251):103-7; Mineharu et al., Mol Cancer Ther. 2014 Sep. 25. pii:molcanther.0400.2014; and Berezhnoy et al., Oncoimmunology. 2014 May 14;3: e28811). Immunotherapies can also include administering one or moreof: adoptive cell transfer (ACT) involving transfer of ex vivo expandedautologous or allogeneic tumor-reactive lymphocytes, e.g., dendriticcells or peptides with adjuvant; cancer vaccines such as DNA-basedvaccines, cytokines (e.g., IL-2), cyclophosphamide, anti-interleukin-2Rimmunotoxins, and/or Prostaglandin E2 Inhibitors (e.g., using SC-50). Insome embodiments, the methods include administering a compositioncomprising tumor-pulsed dendritic cells, e.g., as described inWO2009/114547 and references cited therein. See also Shiao et al., Genes& Dev. 2011. 25: 2559-2572.

Unless explicitly defined elsewhere, the following definitions apply inthe present application.

A “biocompatible polymer” is used here to refer to a polymer that doesnot typically induce an adverse response when inserted or injected intoa living subject, for example, without significant inflammation and/oracute rejection of the polymer by the immune system, for instance, via aT-cell response.

A “copolymer” herein refers to more than one type of repeat unit presentwithin the polymer defined below.

“Encapsulation efficiency” (EE) as used herein is the fraction ofinitial drug that is encapsulated by the nanoparticles (NPs).

“Loading” as used herein refers to the mass fraction of encapsulatedagent in the NPs.

A “polymer,” as used herein, is given its ordinary meaning as used inthe art, i.e., a molecular structure including one or more repeat units(monomers), connected by covalent bonds. The polymer may be a copolymer.The repeat units forming the copolymer may be arranged in any fashion.For example, the repeat units may be arranged in a random order, in analternating order, or as a “block” copolymer, i.e., including one ormore regions each including a first repeat unit (e.g., a first block),and one or more regions each including a second repeat unit (e.g., asecond block), etc. Block copolymers may have two (a diblock copolymer),three (a triblock copolymer), or more numbers of distinct blocks.

A “polymeric conjugate” as used herein refers to two or more polymers(such as those described herein) that have been associated with eachother, usually by covalent bonding of the two or more polymers together.Thus, a polymeric conjugate may comprise a first polymer and a secondpolymer, which have been conjugated together to form a block copolymerwhere the first polymer is a first block of the block copolymer and thesecond polymer is a second block of the block copolymer. Of course,those of ordinary skill in the art will understand that a blockcopolymer may, in some cases, contain multiple blocks of polymer, andthat a “block copolymer,” as used herein, is not limited to only blockcopolymers having only a single first block and a single second block.In some embodiments, the polymeric conjugate is amphiphilic, for exampleby conjugating a hydrophilic polymer, or a cationic/anionic lipid to ahydrophobic polymer.

As used herein, the term “amphiphilic” refers to a property where amolecule has both a polar portion and a non-polar portion. Often, anamphiphilic compound has a polar head attached to a long hydrophobictail. In some embodiments, the polar portion is soluble in water, whilethe non-polar portion is insoluble in water. In addition, the polarportion may have either a formal positive charge, or a formal negativecharge. Alternatively, the polar portion may have both a formal positiveand a negative charge, and be a zwitterion or inner salt. Theamphiphilic compound can be, but is not limited to, one or a pluralityof the following: naturally derived lipids, surfactants, or synthesizedcompounds with both hydrophilic and hydrophobic moieties.

A “particle” refers to any entity having a diameter of less than 10microns (μm). Typically, particles have a longest dimension (e.g.,diameter) of 1000 nm or less. In some embodiments, particles have adiameter of 300 nm or less. Particles include microparticles,nanoparticles, and picoparticles. In some embodiments, particles can bea polymeric particle, non-polymeric particle (e.g., a metal particle,quantum dot, ceramic, inorganic material, bone, etc.), liposomes,micelles, hybrids thereof, and/or combinations thereof. As used herein,the term “nanoparticle” refers to any particle having a diameter of lessthan 1000 nm. In preferred embodiments, a nanoparticle is a polymericparticle that can be formed using a solvent emulsion, spray drying, orprecipitation in bulk or microfluids, wherein the solvent is removed tono more than an insignificant residue, leaving a solid (which may, ormay not, be hollow or have a liquid filled interior) polymeric particle,unlike a micelle whose form is dependent upon being present in anaqueous solution.

“Hydrogel” refers to a substance formed when an organic polymer (naturalor synthetic) is cross-linked via covalent, ionic, or hydrogen bonds tocreate a three-dimensional open-lattice structure which entraps watermolecules to form a gel. Biocompatible hydrogel refers to a polymerforms a gel which is not toxic to living cells, and allows sufficientdiffusion of oxygen and nutrients to the entrapped cells to maintainviability.

As used herein, the term “carrier” or “excipient” refers to an organicor inorganic ingredient, natural or synthetic inactive ingredient in aformulation, with which one or more active ingredients are combined.

As used herein, the term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredients.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” means a dosage sufficient to alleviate one or moresymptoms of a disorder, disease, or condition being treated, or tootherwise provide a desired pharmacologic and/or physiologic effect. Theprecise dosage will vary according to a variety of factors such assubject-dependent variables (e.g., age, immune system health, etc.), thedisease or disorder being treated, as well as the route ofadministration and the pharmacokinetics of the agent being administered.

The term “modulate” as used herein refers to the ability of a compoundto change an activity in some measurable way as compared to anappropriate control. As a result of the presence of compounds in theassays, activities can increase or decrease as compared to controls inthe absence of these compounds. Preferably, an increase in activity isat least 25%, more preferably at least 50%, most preferably at least100% compared to the level of activity in the absence of the compound.Similarly, a decrease in activity is preferably at least 25%, morepreferably at least 50%, most preferably at least 100% compared to thelevel of activity in the absence of the compound.

The terms “inhibit” and “reduce” means to reduce or decrease in activityor expression. This can be a complete inhibition or reduction ofactivity or expression, or a partial inhibition or reduction. Inhibitionor reduction can be compared to a control or to a standard level.Inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

As used herein, the term “prevention” or “preventing” means toadminister a composition to a subject or a system at risk for or havinga predisposition for one or more symptom caused by a disease or disorderto cause cessation of a particular symptom of the disease or disorder, areduction or prevention of one or more symptoms of the disease ordisorder, a reduction in the severity of the disease or disorder, thecomplete ablation of the disease or disorder, stabilization or delay ofthe development or progression of the disease or disorder.

The terms “sufficient” and “effective”, as used interchangeably herein,refer to an amount (e.g. mass, volume, dosage, concentration, and/ortime period) needed to achieve one or more desired result(s).

The term “protein” “polypeptide” or “peptide” refers to a natural orsynthetic molecule comprising two or more amino acids linked by thecarboxyl group of one amino acid to the alpha amino group of another.

The term “polynucleotide” or “nucleic acid sequence” refers to a naturalor synthetic molecule comprising two or more nucleotides linked by aphosphate group at the 3′ position of one nucleotide to the 5′ end ofanother nucleotide. The polynucleotide is not limited by length, andthus the polynucleotide can include deoxyribonucleic acid (DNA) orribonucleic acid (RNA).

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Materials and Methods

The following materials and methods were used in the Examples below.

Materials.

Ester-terminated poly(D,L-lactide-co-glycolide) (PLGA, viscosity0.26-0.54 dL/g) was purchased from Durect Corporation. Cationicethylenediamine core-poly(amidoamine) (PAMAM) generation 0 dendrimer(G0), bafilomycin A1 (Baf A1) were purchased from Sigma-Aldrich. FilipinIII, chlorpromazine (CPZ) and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA)were purchased from Cayman Chemicals (Ann Arbor, Mich., USA). DSPE-PEG(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy{polyethylene glycol}]) with PEG molecular weight (MW) 5000, andceramide-PEG (N-palmitoyl-sphingosine-1-(succinyl{methoxy[polyethyleneglycol]}) with PEG MW of 2000 were obtained from Avanti Polar Lipids.Lipofectamine 2000 (L2K) was purchased from Invitrogen. EGFP mRNA (EGFPmRNA; modified with 5-methylcytidine and pseudouridine) and Cyanine 5fluorescent dye-labeled EGFP mRNA (Cy5 EGFP mRNA; modified with5-methylcytidine and pseudouridine modification) were purchased fromTriLink Biotechnologies. Sequence-verified human PTEN wildtype, G129E,and G129R open reading frame were cloned into pENTR223 followed byrecombination into the Gateway destination vector pHAGE(MSCV-N-Flag-HA-IRES-PURO, long terminal repeat [LTR]-driven expression)using λ recombinase. pLenti CMV Puro LUC (W168) was a gift from EricCampeau (Addgene plasmid #17477)⁷². ViraPower Lentiviral packaging mixwas purchased from Thermo Fisher Scientific. D-luciferin-K+ saltbioluminescent substrate (#122799) was obtained from PerkinElmer.Primary antibodies used in this work included the following: anti-PTEN(138G6), anti-p-Akt-ser473 (#9271), anti-p-70S6K-Thr389 (108D2),anti-p-FOXO3a-Ser318/321 (#9465), anti-p-PARS40-Thr246 (D4D2),p-4E-BP1-Thr37/46 (236B4), p-4E-BP1-Ser65 (#9451) and anti-PARP (#9542)antibodies (rabbit, Cell Signaling); anti-HA antibody (3F10) (rat,Roche); anti-HA-HRP conjugated antibody (A00169) (goat, GenScript);Anti-GFP antibody (A-6455) (rabbit, Life Technologies).

Preparation of Modified PTEN mRNA.

Vector carrying open-reading frame (ORF) of PTEN was a gift from WilliamSellers⁷³ (pSG5L HA PTEN wt; Addgene #10750). The vector was linearizedby Apal/EcoRl digestion and purified. HA-PTEN ORF under the regulationof T7 promoter was then amplified by PCR reaction. The amplicons werefurther purified and used as templates for in vitro transcription (IVT).The modified PTEN-mRNA was synthesized as described previously^(48, 49)In brief, IVT was conducted using MEGAscript T7 kit (Ambion) with 1-2 μgtemplate and 7.5 mM ATP, 1.5 mM GTP, 7.5 mM 5-methyl-CTP, 7.5 mMpseudo-UTP (TriLink Biotechnologies), and 6 mM 3′-0-Me-m⁷G(5′)ppp(5′)G(anti-reverse cap analog, ARCA) (TriLink Biotechnologies). Reactionswere incubated at 37° C. for 4 hours, followed by Turbo DNase treatmentfor 15 min. 3′ poly(A)-tails were further added to IVT RNA productsusing a poly(A) tailing kit (Ambion). mRNA was purified by using theMEGAclear kit (Ambion), then treated with Antarctic Phosphatase (NewEngland Biolab) at 37° C. for 30 min, and further purified. Large-scalePTEN mRNA was custom-prepared by TriLink Biotechnologies as above (ARCAcapped and enzymatically polyadenylated; fully substituted with Pseudo-Uand 5′-Methyl-C; DNase and phosphatase treatment; Silica membranepurification) using 100-150 μg template containing T7 promoter andHA-PTEN ORF.

Synthesis of Cationic Lipid Compound (G0-C14).

The cationic lipid-like compound (G0-C14) was synthesized fromethylenediamine core-poly(amidoamine) (PAMAM) generation 0 dendrimer(G0) using a ring-opening reaction by reacting with 1,2 epoxytetradecaneaccording to previously described procedure^(45, 74). Briefly, 1,2epoxytetradecane was mixed with PAMAM dendrimers G0 at a molar ratio of7:1, where substoichiometric amounts of 1,2 epoxytetradecane were addedto increase the proportion of products with one less tail than the totalpossible for a given amine monomer. The reaction was carried out for 2days under vigorous stirring, and the crude mixture was separated onsilica with gradient elution from CH₂Cl₂ to 75:22:3 CH₂Cl₂/MeOH/NH₄OHusing chromatography.

mRNA Complexation Ability of G0-C14 and its Stability in OrganicSolvent.

To assess the mRNA complexation ability of G0-C14 and its stability inorganic solvent (DMF), naked EGFP-mRNA or EGFP-mRNA complexed withG0-C14 (in varying weight ratios from 1 to 20) were incubated with orwithout DMF for 30 min. For mRNA samples in DMF, electrophoresis was runwithout extracting mRNA from DMF into aqueous solution. The volumes ofsamples were then adjusted with loading dye (Invitrogen) and run into anE-Gel® 2% agarose (Invitrogen) gel for 30 min at 50 V. The Ambion®Millennium™ markers-formamide (Thermo Fisher Scientific) was used as aladder. Finally the gel was imaged under UV and the bands were analyzed.

Preparation of mRNA NP.

We employed a robust, innovative self-assembly method to preparemRNA-encapsulated polymer-lipid hybrid NPs as we previously described⁴⁵,but with significant modification and optimization in ratios of reagentsused in NP formulation. In brief, PLGA and G0-C14 were dissolvedseparately in dimethylformamide (DMF) at concentrations of 5 mg/ml and2.5 mg/ml, respectively. Then PLGA (250 μg in 50 μl) and G0-C14 (250 μgin 100 μl) were mixed at a weight ratio of 1:1 in a small glass vial.mRNA (16 μg at 1 mg/ml concentration) in aqueous solution was mixed intothe PLGA/G0-C14 organic solution (weight ratio of mRNA:PLGA:G0-C14 was1:15:15) to form cationic lipid/mRNA nanocomplexes. This solution wasthen quickly nanoprecipitated into 10 ml of lipid-PEG (e.g.,ceramide-PEG or DSPE-PEG) aqueous solution (0.1 mg/ml concentration inDNase/RNase-free Hypure water) for ˜20 seconds. The weight ratio oflipid-PEG to PLGA was 4:1. Upon nanoprecipitation, NPs formed instantlyand were kept for 30 min at 600 rpm stirring at room temperature tostabilize. The NPs were then washed three times with ice-cold Hypurewater using Amicon tubes (MWCO 100 kDa; Millipore) to remove organicsolvent and free compounds and finally concentrated into 1 ml PBSsolution. The NPs were used fresh or kept at −80° C. to use later forvarious in vitro and in vivo studies. The mRNA NPs were run through gelelectrophoresis as described above to check for any unencapsulated mRNAleaching. The NPs prepared with ceramide-PEG and DSPE-PEG were termedPGCP and PGDP NPs, respectively.

Physicochemical Characterization and Stability of mRNA NPs in SerumCondition.

mRNA NPs were characterized by assessing their size, surface charge, andmorphology. Sizes were measured by NanoSIGHT (Malvern, NS300) at 20° C.and analyzed using Nanoparticle Tracking Analysis (NTA), which utilizesthe properties of both light scattering and Brownian motion to determinethe particle size distribution of samples in liquid suspension. Thesurface charge of the NPs was determined by dynamic light scattering(DLS) with 15-mW laser and an incident beam of 676 nm (BrookhavenInstrument Corporation). A transmission electron microscope (TEM) wasused to assess the NPs' morphology and shape. For TEM, NPs were stainedwith 1% uranyl acetate and imaged using a Tecnai G² Spirit BioTWINmicroscope (FEI Company) at 80 kV. To check the in vitro stability ofpolymer-lipid hybrid mRNA NPs in serum as a means to mimic in vivoconditions, mRNA NPs were incubated in 10% serum containing PBS solutionat 37° C. in triplicate for various time periods (0, 2, 4, 8, 12, 24,and 48 h) with 100 rpm shaking. At each time point, an aliquot of NPssolution was taken for particle size measurement using NanoSIGHT andanalyzed as described above to evaluate any change in size distributionat various time intervals. EGFP mRNA NPs were used in this study.

Cell Culture.

Human PCa cell lines (PC3, DU145, LNCaP and its invasive subclone LNCaPLN3) and prostate epithelial cells (PreC) along with two breast cancercell lines (MDA-MB-468 and MDA-MB-231) were used in various in vitrostudies. All cells were purchased from American Type Culture Collection(ATCC). Cells were maintained in F-12K (ATCC), Eagle's Minimum EssentialMedium (EMEM; ATCC), Roswell Park Memorial Institute (RPMI) 1640 (ATCC),or Leibovitz's L-15 (ATCC) cell-culture medium, according to the culturemethod for each cell type per the instructions from ATCC, supplementedwith high-glucose, 10% fetal bovine serum (FBS; Gibco®) and 1%penicillin/streptomycin antibiotic (Thermo-Fisher Scientific). Cellculture and all biological experiments were performed at 37° C. in 5%CO₂ conditions in a cell-culture incubator. All cells were authenticated(using the “DDC Biomedical” or “Genetica DNA Laboratories” cell lineauthentication test) and checked for mycoplasma contamination before invitro cell experiments and in vivo xenograft tumor model preparation.

Generation of Luciferase-Tagged PC3 Cells.

The lentiviral vector pLenti CMV Puro LUC encoding the fireflyLuciferase was transfected with Virapower Lentiviral packing mix to 293Tcells using lipofectamine 2000. After 48 h, lentiviral supernatant wascollected and added into 20-40% confluent PC3 cells. Polybrene (8 μg/ml)was added during the transduction. Two days after transduction, PC3cells were selected by puromycin at 2 μg/ml concentration. Luciferaseexpression was analyzed by immunofluorescence staining and western blot.PC3-luc cells were maintained in media containing 1 μg/ml puromycin.

In Vitro Cytotoxicity and Transfection Activity of mRNA NP.

Cells were seeded at a density of 3-5×10⁴ cells per well on 24-wellplate and allowed to attach and grow until ˜80% confluence. Cells weretransfected with mRNA NPs at various mRNA concentrations (0.062, 0.125,0.250, and 0.500 μg/ml) for 16 h followed by washing with fresh completemedium and further incubated for 24 h to check cytotoxicity as well astransfection efficiency. Lipofectamine 2000 (L2K) was used as a standardtransfection reagent (according to manufacturer's protocol) to formL2K-mRNA complexes for comparison with the mRNA NPs. Cytotoxicity wasmeasured by AlamarBlue® assay according to the manufacturer's protocolusing a microplate reader (TECAN, Infinite M200 Pro). AlamarBlue is anon-toxic assay that allowed us to continuously check real-time cellproliferation. For transection efficiency measurement, cells wereharvested with 25% EDTA trypsin and washed two times and resuspended inPBS followed by measuring GFP expression using flow cytometry. Thepercentages of GFP-positive cells were calculated, and histograms wereprepared using Flowjo software.

RNase Protection Assay.

To test whether the NPs protected the mRNA from RNase, naked EGFP mRNAand EGFP-mRNA PGCP NPs were incubated in RNase at two mRNA-to-RNaseweight ratios (1:1 and 1:10) for 30 min at 37° C., shaking at 100 rpm. Aconcentration of 0.250 μg/ml EGFP mRNA was used in this study. Afterincubation, RNase was separated from the EGFP-mRNA PGCP NPs and nakedmRNA by washing with water via centrifugation in 100 kDa Amicon filtertubes at 1300 rcf for 10 min. The post RNase-treated EGFP-mRNA PGCP NPswere then diluted in media, while the naked mRNA was complexed with L2K,and the PC3 cells were then transfected as described above and incubatedfor 16 h. The medium was replaced and incubated for an additional 24 h.The naked EGFP mRNA and EGFP-mRNA PGCP NPs without RNase treatment wereused as negative controls. Cells were then harvested to measure EGFPexpression by flow cytometry and analyzed using Flowjo as describedabove.

Mechanism of Cellular Uptake and Endosomal Escape of mRNA NPs.

To determine the mRNA NPs' uptake and intracellular transport mechanism,24-well plates were used to seed PC3 cells at an initial density of5×10⁴ cells/ml in 1 ml of growth medium and incubated for 24 h at 37° C.in 5% CO₂ to allow the cells to attach. The cells were thenpre-incubated for 30 min in serum-free medium containing inhibitors(Filipin at 1 μg/ml, CPZ at 10 μg/ml, EIPA at 10 μg/ml, and Baf A1 at200 nM was used to block caveolae-mediated endocytosis,clathrin-mediated endocytosis, macropinocytosis, and intracellularproton pump inhibitor effects, respectively, alone and in combination).The cells were then transfected with EGFP-mRNA PGCP NPs at mRNAconcentration of 0.250 μg/ml. After 16 h, the old medium was replacedwith fresh complete medium and incubated for an additional 24 h. Thecells were then harvested to check EGFP expression by flow cytometry andanalyzed by Flowjo as described above.

Cell Growth Inhibition Assay with PTEN mRNA NP.

Cell growth inhibition was determined by CyQUANT assay in 96-wellplates. First, 3-5×10³ cells per well per 100 μl were seeded in 96-wellplates. The next day, cells were treated with mRNA NP or the L2K-mRNAmixture. After 72 h incubation under standard cell-culture conditions,the culture medium was removed and plates were kept at −80° C. for >24h. Cells were counted using the CyQUANT kit (Life Technologies) per themanufacturer's instructions. Fluorescence measurements were made using amicroplate reader with excitation at 485 nm and emission detection at530 nm. Cell-growth inhibition was also determined using a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromideassay. Briefly, 3-5×10³ cells were plated in a 96-well plate and treatedwith NPs the next day. After 16 h, NPs were removed and fresh medium wasadded. After 72 h of incubation, 10% culture volume of MTT (SigmaChemicals, St. Louis, Mo.; 5 mg/ml) was added to each well. Afterincubation for an additional 4 h, 200 μl of isopropanol-HCl solution wasadded to each well to dissolve the cell pellets. Absorbance wasdetermined using a 96-well SpectraMax plate reader (Molecular Devices,Sunnyvale, Calif.) at 560 nm and 650 nm (background).

Apoptosis Assay In Vitro.

Cells were seeded in 6-well plates until ˜80% confluence and thentreated with PTEN mRNA NPs. The next day, the NPs were removed (16 hpost treatment) and kept in culture for another 24 h. The supernatantand the cell monolayer were collected, washed with PBS, and processedfor detection of apoptotic cells using the Annexin V-PE/7AAD apoptosisdetection kit (BD Biosciences) according to the manufacturer'sinstructions.

Western Blot Assay.

Protein extracts were prepared using NP-40 lysis buffer (50 mM Tris-HCl[pH 7.5], 0.5% NP-40 substitute, 150 mM NaCl, and 12.5 mM NaF)supplemented with Complete Mini EDTA-free protease inhibitor tablets(Roche). Equal amounts of protein, as determined with a bicinchoninicacid (BCA) protein assay kit (Pierce/Thermo Scientific) according to themanufacturer's instructions, were separated by SDS-PAGE and transferredto nitrocellulose membranes. The blots were blocked with 5% non-fat drymilk in TBST (50 mM Tris-HCl at pH 7.4 and 150 mM NaCl, and 0.1% Tween20) and then incubated with appropriate primary antibodies. Signals weredetected with horseradish peroxidase-conjugated secondary antibodies andan enhanced chemiluminescence (ECL) detection system (Amersham/GEHealthcare). When indicated, membranes were subsequently stripped forreprobing.

Immunofluorescent Staining and Microscopy.

For immunofluorescent staining, cells were plated onto coverslips in6-well plates and grown overnight to 60-70% confluence. Cells werewashed with ice-cold PBS and fixed with 4% paraformaldehyde (PFA,Electron Microscopy Sciences) in PBS for 15 min at room temperature(RT). Cells were then permeabilized by incubation in 0.2% TritonX-100-PBS for 8 min followed by blocking with PBS blocking buffercontaining 2% normal goat serum, 2% BSA, and 0.2% gelatin for 1 h at RT.Then the samples were incubated in primary antibody (1:200 anti-HA ratantibody) for 1 h at RT, washed with PBS, and incubated in goatanti-rat-Alexa Fluor 488 (Molecular Probes) at 1:500 dilution inblocking buffer for 30 min at RT. Finally, stained cells were washedwith PBS, counterstained with 500 nM DAPI, and mounted on slides withProlong Gold antifade mounting medium (Life Technologies).

Animals.

Six-week-old BALB/c male normal mice were used for pharmacokinetics (PK)and immune response studies. To evaluate the biodistribution (BioD) ofmRNA NP in various organs including tumors and test the therapeuticefficacy of PTEN mRNA NP to suppress tumor growth, male athymic nudemice (6 weeks old) were obtained from Charles River Laboratories. Allanimal studies were performed under strict regulations and pathogen-freeconditions in the animal facility of Brigham and Women's Hospital and inaccordance with National Institutes of Health animal care guidelines.The animals had free access to sterile food pellets and water and werekept in the laboratory animal facility with temperature and relativehumidity maintained at 23±2° C. and 50±20%, respectively, under a 12-hlight/dark cycle. Mice were kept for at least one week to acclimatizethem to the food and environment of the animal facility. The animalprotocol was approved by the Institutional Animal Care and UseCommittees at Harvard Medical School.

Pharmacokinetic (PK) Study.

For in vivo PK study, healthy BALB/c male mice (6 weeks) were dividedinto three groups (n=3 per group) and intravenously administered (i)naked Cy5 EGFP mRNA, (ii) Cy5-EGFP-mRNA-PGCP NP or (iii)Cy5-EGFP-mRNA-PGDP NP through the tail vein at a mRNA dose of 700 μg perkg of animal weight. At various predetermined time intervals (0, 5, 15,30, 60, 120, 180, and 240 min), retro-orbital vein blood was withdrawnusing a heparin-coated capillary tube, and the wound was gently pressedfor a few seconds to stop the bleeding. Fluorescence intensity of Cy5was measured at emission and excitation wavelengths of 640 and 670 nm,respectively, using a microplate reader. PK was calculated bycalculating the percentage of Cy5 EGFP mRNA in blood at various timeperiods, normalized with the initial (0 min) time point.

PC3-Xenograft Tumor Model Preparation.

To prepare the PC3-xenograft tumor mice model, about 4×10⁶ cells in 100μL of culture medium mixed with 100 μL of matrigel (BD Biosciences) wereimplanted subcutaneously on the right flank of 6-week-old male athymicnude mice. Mice were monitored for tumor growth every other dayaccording to the animal protocol.

Biodistribution (BioD) of mRNA NP in PCa Xenograft Tumor Model.

For the BioD study, PC3 xenograft-bearing male athymic nude micereceived intravenous injection of naked Cy5 EGFP mRNA,Cy5-EGFP-mRNA-PGCP NP, and Cy5-EGFP-mRNA-PGDP NP via tail vein injectionat an mRNA dose of 700 μg per kg of animal weight. Twenty-four hourslater, organs and tumors were harvested and imaged with the IVIS LuminaIII In Vivo Imaging System (Perkin Elmer). To evaluate in vivo BioDspecifically for PTEN mRNA in a tumor xenograft model, Cy5-tagged PTENmRNA was prepared by substitution of 25% psedu-UTP with Cy5-UTP whenconducting IVT reactions. The Cy5-PTEN-mRNA and its PGCP and PGDP NPswere then injected (i.v. via tail vein) for BioD analysis as describedabove.

In Vivo Therapeutic Efficacy of PTEN mRNA NP in PCa Xenograft TumorModel.

For in vivo therapeutic efficacy, PGDP NP was used as a delivery systemfor PTEN mRNA and EGFP mRNA as a negative control. PC3 xenograft-bearingathymic nude mice were treated when the tumors were first palpable. Themice were randomly divided into three groups, which received (i) PBS(n=7), (ii) EGFP-mRNA-PGDP NP (n=9), or (iii) PTEN-mRNA-PGDP NP (n=8).Mice were injected with the above samples via tail vein at a mRNA doseof 700 μg per kg of animal weight at days 10, 13, 16, 19, 22, and 25after tumor induction. Tumor size was measured using a caliper everythree days through day 43, and average tumor volume (mm³) was calculatedas: ½(length×width×height). The body weights of the mice were alsodetermined. At day 28 (three days after the last injection), mice (1mouse for PBS and 2 mice for each EGFP-mRNA-PGDP NP and PTEN-mRNA-PGDPNP group) were selected randomly for harvest of tumors to monitor PTENexpression and tumor cell apoptosis, and various organs to examine invivo toxicity. The mice were imaged at day 35, and the image backgroundswere removed using Adobe Photoshop software. At day 43, mice weresacrificed and various organs (lung, heart, liver, kidney, and spleen)were collected to assess toxicity by immunohistochemical analysis. Bloodserum was also collected at the two time points of days 28 and 43 forhematological assays.

In Vivo Therapeutic Efficacy of PTEN mRNA NPs in Advanced PCa Models.

To assess the in vivo therapeutic efficacy of PTEN mRNA NP in advancedPCa, we prepared two different advanced PCa mice models: (1) anexperimental metastasis model employing intravenous inoculation ofluciferase-expressing PC3 (PC3-luc) prostate cancer (PCa) cells, and (2)a bone colonization of intratibial (IT) inoculation of PC3-luc cells asan orthotopic model of PCa established metastases. For experimentalmetastatic PCa model, 2.5×10⁶PC3-luc cells in 100 μL of PBS wereimplanted through i.v. tail vein injection into immunocompromised, maleathymic nude mice (78 in total). Two weeks after implantation, mice weremonitored for tumor growth every three days using an In-Vivo Xtremeimaging system (Bruker) for initial screening of PC3-luc disseminatedmice for treatments. Four weeks post tumor inoculation, 24 mice(incidence rate of ˜30%) with i.v. disseminated PCa (detected using anIn-Vivo Xtreme imaging system [Bruker] after mice were injectedintraperitoneally with 150 mg/kg luciferin substrate [PerkinElmer,Catalog #122799]) were randomly divided into three groups (n=8 pertreatment group), which received (i) PBS, (ii) EGFP-mRNA-PGDP NP, or(iii) PTEN-mRNA-PGDP NP. To prepare IT orthotopic PCa model,immunocompromised, male athymic nude mice were anesthetized withisoflurane, and 5×10⁴ PC3-luc cells in 10 μL of PBS were injected intibiae of each mouse (40 mice [80 tibae] in total). Note that theestablishment of IT orthotopic PCa model had a ˜90% success rate. Themice were then randomly divided into the above three groups (n=12 mice[n=24 tibae] per treatment group) and treatments were started next day(considered as day 0) post tumor cell inoculation. Treatments wereperformed via i.v. tail vein injection at mRNA dose of 700 μg per kg ofanimal weight. Initial treatment was performed at day 0, followed byanother four injections in every three days (in total, five injections).Tumor images were also obtained every three days using an In-Vivo Xtremeimaging system (Bruker) as mentioned above, using a charge-coupleddevice (CCD) camera (exposure time 30 sec, binning of 1, field of vision[FOV] of 19 cm, f/stop of 1.10, and no filter). Regions of interest(ROI) were quantified as average radiance (photon/sec/cm²/sr) usingBruker MI SE software, and the fold change of ROI in each measurement(day 3, 6, 9, 12 and 15) was compared to day 0 of the same tibia andplotted using GraphPad software (Version 7). The body weights of themice were also determined every three days and plotted using GraphPadsoftware (Version 7).

Immunohistochemical Staining to Detect In Vivo PTEN Expression.

The expression of HA-PTEN protein in tumor tissue section was assessedby immunohistochemistry. Sections (5 μm thick) were obtained from tumorstreated with PBS, PTEN-mRNA-PGDP NP, or EGFP-mRNA-PGDP NP.Paraffin-embedded sections were deparaffinized, rehydrated, and washedin distilled water. Samples were then incubated for 20 min with 0.3%hydrogen peroxide (H₂O₂) at room temperature to quench endogenousperoxidase activity followed by antigen retrieval in citrate buffer (pH6.0) using a microwave for 10 min (2 times, each time 5 min). Afterwashing with PBS (pH 7.4), the samples were treated with theAvidin/Biotin Blocking kit (Vector) to quench endogenous biotin, andthen immersed in blocking buffer (1% BSA, 5% normal goat serum) for 60min. Tissue sections were then incubated with primary rabbit anti-HAantibody at 4° C. overnight in a humid chamber. After being rinsed withPBS, the samples were incubated with biotinylated secondary antibody for30 min at room temperature, followed by incubation with theavidin-biotin-horseradish peroxidase complex (ABC kit, VectorLaboratories, Inc). Staining was developed with the diaminobenzidineperoxidase substrate kit (Impact DAB, Vector Laboratories, Inc) for 3min. Sections were then counterstained with hematoxylin (Sigma),dehydrated, and mounted.

TUNEL Apoptosis Assay.

Tumors were extracted and fixed in formalin, embedded in paraffin, andsectioned at a thickness of 5 μm. Tumor cell apoptosis was determined byterminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphatenick-end labeling (TUNEL) assay (In Situ Cell Death Detection Kit, TMRred; Roche, #12-156-792-910) according to the manufacturer's protocol.DAPI stain was used to assess total cell number.

In Vivo Toxicity Evaluation: Hematologic Examination, Histology, andImmune Response.

To evaluate in vivo toxicity, blood was drawn retro-orbitally and serumwas isolated from PC3 xenograft athymic nude mice three days after thefinal injection (day 28), and at the end of the efficacy experiment (day43). Aspartate aminotransferase (AST), alanine aminotransferase (ALT),blood urea nitrogen (BUN), creatinine, and troponin-1 were measuredusing assay kits for AST (BioVision), ALT and Creatinine (CaymanChemical), BUN (Arbor Assays), and troponin-1 (Life Diagnostics)according to the manufacturers' protocols. For histological examination,various organs (lung, heart, liver, kidney, and spleen) were alsocollected three days after the final NP injection (day 28), and at theend point of the experiment (day 43). The organs were then fixed with 4%paraformaldehyde and embedded in paraffin followed by sectioning (3˜4μm) and staining with H&E. The slides were assessed using an EVOS CellImaging System (Thermo Fisher Scientific). Next, to check immunologicalresponse, male Balb/c immunocompetent male mice (n=3) receivedintravenous injection of PBS, naked PTEN mRNA (700 μg mRNA per kg),empty PGDP NP, and PTEN-mRNA PGDP NP. Six or twenty-four hours postinjection, serum samples were collected and processed to measure therepresentative cytokine (i.e., TNF-α) by enzyme-linked immunosorbentassay (ELISA) (Affymetric eBioscience) according to the manufacturer'sprotocol.

Statistical Analysis.

All graphs were prepared using GraphPad Prism 7 software, andstatistical analysis was also carried out using GraphPad Prism 7software to perform One-Way ANOVA or Mann-Whitney test Mann-Whitneytests were performed for experiments in which the data was determined tobe nonparametric by the normality test⁷⁵⁻⁷⁷ (i.e., for both disseminatedmetastatic and IT orthotopic experiments). All experiments wereperformed in triplicate unless otherwise stated. Error bars indicatestandard deviation (SD), unless otherwise noted specifically as standarderror means (SEM). A P<0.05 value is considered statisticallysignificant, where all statistically significant values shown in variousFigures are indicated as: * P<0.05, **P<0.01, ***P<0.001, and****P<0.0001.

Example 1. Preparation and Characterization of mRNA NP

A robust self-assembly approach was employed to prepare the hybrid mRNANPs using the cationic lipid-like compound G0-C14 andpoly(lactic-co-glycolic acid) (PLGA) polymer coated with alipid-poly(ethylene glycol) (lipid-PEG) shell⁴⁵ (FIG. 1A). G0-C14 wasused for mRNA complexation, and PLGA, a widely clinically usedbiodegradable and biocompatible polymer, was used to make a stable NPcore. EGFP mRNA was used as a model mRNA, and the EGFP mRNA NP coatedwith ceramide-PEG is herein referred as to EGFP-mRNA-PGCP NP. Weobserved no effect of organic solvent (DMF) on the integrity orstability of EGFP mRNA, whether naked, complexed with G0-C14, orencapsulated in NPs (FIG. 1B). FIG. 1B also shows that G0-C14effectively condensed EGFP mRNA at a weight ratio of 5 or above. The NPswere prepared at a G0-C14/mRNA weight ratio of 15, with no leaching ofmRNA shown by electrophoresis, suggesting that most mRNA wasencapsulated. The hybrid EGFP-mRNA-PGCP NPs were ˜120 nm in size andspherical, as characterized by NanoSIGHT and transmission electronmicroscopy (TEM), respectively (FIG. 1C). Essential to the hybrid NP,the solid PLGA polymer core allowed formation of a stable and rigidnanostructure. The average surface charge measured by dynamic lightscattering (DLS) was near neutral (5.96±0.76 mV), since the NPs have anouter lipid-PEG shell. In addition, the serum stability test showed noobvious changes in the particle size over 48 h, suggesting the stabilityof the EGFP-mRNA-PGCP NPs (FIG. 1D).

Example 2. mRNA NPs Exhibit Low Cytotoxicity and Potent TransfectionEfficiency In Vitro, and Protect mRNA Activity from RNase Degradation

To evaluate in vitro cytotoxicity, cells were treated withEGFP-mRNA-PGCP NPs for 16 h and further incubated with fresh culturemedium for 24 h; nearly 80% of PC3 cells were still viable at thehighest EGFP mRNA concentration of 0.5 μg/ml (FIG. 2a ). AlamarBluetoxicity assay was further extended for DU145 and LNCaP cells with nonotable reduction in cell viability, maintaining ˜90-100% viable cellsat various EGFP mRNA concentrations from 0.062 to 0.5 μg/ml in DU145cells and 100% cell viability at all concentrations in LNCaP cells (FIG.9A and FIG. 10A, respectively).

We next examined transfection efficacy in vitro. The EGFP-mRNA-PGCP NPmediated highly efficient transfection of PC3 cells, showing adose-dependent linear increase of EGFP expression correlated withincreasing EGFP mRNA concentrations (from 0.062 to 0.5 μg/ml) (FIG.2B,C). The transfection efficacy (in terms of percentile ofEGFP-positive cells) of the NPs at mRNA concentrations of 0.25 and 0.5μg/ml was demonstrably greater than that mediated by the commercialtransfection agent lipofectamine 2000 (L2K) at mRNA concentration of 0.5μg/ml. This high transfection activity was confirmed by confocalmicroscopy (FIG. 2D), although L2K-mRNA-transfected PC3 cells exhibiteda slightly higher fluorescent intensity. We found similar highlyeffective transfection activity of EGFP-mRNA-PGCP NPs in two other PCacell lines (DU145 and LNCaP); >98% and 86% efficiency at the 0.5 μg/mlconcentration, respectively (FIG. 9B,C and FIG. 10B,C). While thetransfection activity of our mRNA NP was comparable to that of L2K-mRNAin DU145 cells, the NP group showed significantly greater transfectionefficacy relative to L2K-mRNA in LNCaP cells, especially at EGFP mRNAconcentrations of 0.25 and 0.5 μg/ml.

To investigate the ability of the NPs to protect mRNA from RNasedegradation, we incubated EGFP-mRNA-PGCP NP at two ratios of mRNA toRNase weight (1:1 and 1:10) for 30 min and then evaluated transfectionactivity in PC3 cells. A concentration of 0.250 μg/ml EGFP mRNA wasused. Naked EGFP mRNA (without or with RNase incubation) complexed withL2K was used as a control. The transfection of naked EGFP mRNA (withoutRNAse incubation) complexed with L2K showed ˜85% efficiency, whereastransfection was drastically reduced to <0.1% (similar to untreatedcontrol) in the RNase-treated groups. In contrast, EGFP-mRNA-PGCP NPnotably maintained the integrity and activity of the mRNA at both RNaseconcentrations, consistently showing ˜90% transfection capacity,comparable to the transfection in the absence of RNAse (FIG. 11A,B).

Example 3. Mechanisms of Cellular Uptake and Endosomal Escape of mRNANPs

To evaluate the cellular uptake mechanisms and intracellular transportof mRNA NPs, we studied the transfection efficiency of EGFP-mRNA-PGCPNPs in PC3 cells pre-treated with different inhibitors. NP transfectionwas not affected by either caveolae- or clathrin-mediated inhibitors(Filipin and CPZ, respectively). In contrast, uptake was significantlydecreased from ˜80% (without inhibitor) to ˜40% in the presence of EIPA,a macropinocytosis inhibitor. Transfection of mRNA NPs was also markedlydecreased from ˜80% (without inhibitor) to ˜58% in the presence of theproton-pump inhibitor Bafilomycin A1 (Baf A1)^(46, 47). We furthertested transfection activity using combinations of Filipin, CPZ, or EIPAwith Baf A1. Filipin+Baf A1 and CPZ+Baf A1 exhibited low transfectionefficiency similar to Baf A1 alone, whereas EIPA+Baf A1 showed acombinatorial effect, exhibiting superior inhibition of transfectionactivity (FIG. 2E,F). These results suggest that cellularinternalization of the EGFP-mRNA-PGCP NP is partly mediated bymacropinocytosis, and after entering into the cells, the NPs were ableto induce a proton-sponge effect to release the cargo cytosolically. Allthe above inhibitors are used commonly to investigate the cellularuptake/transport pathways of NP⁴⁵⁻⁴⁷. The results thus suggest that thiscombinatorial cellular uptake and endosomal escape mechanism, togetherwith mRNA's inherent properties, could constitute one potentialadvantage of this mRNA delivery system.

Example 4. Functional NP Delivery of PTEN mRNA to PTEN-Null PCa Cells InVitro

We prepared PTEN mRNA by in vitro transcription (IVT) as previouslydescribed^(48, 49) PTEN mRNA was modified with ARCA capping andenzymatic polyadenylation and was fully substituted with Pseudo-UTP,5′-Methyl-CTP, followed by DNase and phosphatase treatment. Substitutionwith Pseudo-UTP and 5′-Methyl-CTP in replacement of regular UTP and CTPwas applied to reduce mRNA immunostimulation.^(43, 49, 50) The PTEN mRNAwas also hemagglutinin (HA)-tagged to ensure easy detection andseparation from endogenous message. We first transfected PTEN mRNA intoPTEN-null PCa (PC3) cells using L2K to assess the facilitation ofprotein expression, diminish cancer cell viability, and suppress thePI3K-AKT pathway. PC3 cells transfected with L2K-PTEN-mRNA showedmarkedly higher PTEN-HA expression than pHAGE-PTEN WT(MSCV-N-Flag-HA-IRES-PURO gateway destination vector with long terminalrepeat [LTR]-driven expression of PTEN wildtype) by immunofluorescencestaining (FIG. 12A). Western blotting also showed that PTEN-HAexpression was significantly higher than plasmid PTEN transfection. Thedifference in Akt-Ser473 level between empty PGCP NP and PTEN-mRNA-PGCPNP was masked by relatively high background levels of Akt-Ser473generated by growth factors contained in fetal bovine serum. However,when compared to the basal level of Akt-Ser473 in serum starvationconditions, there was a significant decrease in Akt-Ser473 levels whenPTEN-mRNA-PGCP NPs were added to induce PTEN expression. Furthermore,PTEN mRNA treatment downregulated the PI3K-AKT pathway, showingdecreased in phosphorylation of 4E-BP1-Ser65, PARS40-Thr246, andFoxo3a-Ser318/321 as determined by western blotting (FIG. 12B).Accordingly, PTEN mRNA NP treatment dramatically decreased cellviability as measured by CyQUANT assay (FIG. 12C).

Next, we applied the NP platform optimized above to determine whetherour PTEN-mRNA-PGCP NP could restore the therapeutic functionality oftumor-suppressor PTEN to PCa cells. Both immunofluorescence staining(FIG. 3A) and western blot (FIG. 3B) confirmed the restoration ofPTEN-HA expression transduced by PTEN-mRNA-PGCP NP treatment. It isworth noting that since PTEN-mRNA-PGCP NP treatment reduced cellviability, the cell density of this group was considerably lower thanthat of the group treated with control empty PGCP NP (FIG. 3A). Next,PTEN-mRNA-PGCP NP treatment significantly decreased cell viability in adose-dependent manner compared to both empty PGCP NP and EGFP-mRNA-PGCPNP groups, as measured by MTT assay (FIG. 3C). We further found thatafter 48 h treatment, PTEN-mRNA-PGCP NP efficiently inhibited PI3K-AKTsignaling as indicated by the greater decrease in phosphorylation ofAkt-Ser473, p70S6K-Thr389, 4E-BP1-Thr37/46, PARS40-Thr246, andFoxo3a-Ser318/321. PTEN-mRNA-PGCP NP treatment also reduced basalphosphorylation of the above proteins under serum-starvation conditions(FIG. 3B). Moreover, early apoptosis was increased after treatment withPTEN-mRNA-PGCP NP, as indicated by elevated numbers ofAnnexin-V-positive cells via flow cytometry (FIG. 3D). A ˜4-foldincrease in cell death was noted in PC3 cells after PTEN-mRNA-PGCP NPtreatment relative to control empty PGCP NP. We also noticed a slightincrease in apoptosis in the EGFP-mRNA-PGCP NP-treated group; this wasin part a possible consequence of inducing exogenous RNA into PC3 cells.However, this effect was very modest in comparison to the increase inapoptosis observed with the PTEN-mRNA-PGCP NP group. These resultsindicate that our mRNA NP platform has the potential to effectivelydeliver PTEN mRNA and restore functional PTEN activity to tumor cells.

Consistent with the above results, PTEN-mRNA-PGCP NP treatmentremarkably reduced the cell viability of androgen receptor (AR)-positivePCa LNCaP cells as well as the invasive LNCaP LN3 subclone, bothPTEN-deficient (FIG. 13A). Cell apoptosis was also increased in LNCaPcells by PTEN-mRNA-PGCP NP treatment relative to empty PGCP NP orEGFP-mRNA-PGCP NP control groups (FIG. 13B). In contrast, delivery ofPTEN-mRNA with our PGCP NP into normal prostate epithelial cells (PreC)or to PTEN-competent DU145 cells did not significantly affect cellviability (FIG. 14A), nor was there any significant change in PI3K-AKTsignaling (FIG. 14B). This is consistent with earlier results⁵¹ showingthat PTEN^(+/−) DU145 was refractory to conventional transfection withPTEN plasmid and suggests that restoration of PTEN may most effectivelysuppress growth and survival of tumor cells with defective PTENexpression. Similar to the results with PCa cells, PTEN-mRNA-PGCP NPtreatment restored PTEN protein and growth-suppressive activity inPTEN-null MDA-MB-468 breast cancer cells, with treated cells showingreduced cell viability and PI3K-AKT signaling as well as inducedapoptosis via PARP cleavage and Annexin V staining. PTEN-competentMDA-MB-231 cells showed no effect on those phenotypes afterPTEN-mRNA-PGCP NP treatment (FIG. 15A-C). Together, our results indicatethe potential of PTEN-mRNA-PGCP NPs to restore the tumor-suppressiveactivity of PTEN in PTEN-defective cells of different tumor origins.

Example 5. In Vivo Pharmacokinetics and Biodistribution of mRNA NPs

To predict the in vivo performance of our mRNA NPs for systemicdelivery, we first evaluated pharmacokinetics (PK) by administeringCy5-EGFP-mRNA NPs prepared with two different lipid-PEGs (ceramide-PEGand DSPE-PEG, termed PGCP and PGDP, respectively) into healthy BALB/cmice via tail-vein intravenous (i.v.) injection and comparing the PKwith that of naked Cy5-EGFP-mRNA. The EGFP-mRNA-PGDP NP possessedexcellent physicochemical properties, with particle size and surfacecharge of 112.7±1.3 nm and 5.22±0.43 mV, respectively (FIG. 16A) alongwith good stability in serum conditions and stable particle size over 48h at 37° C. (FIG. 16B). Notably, the in vitro transfection efficiency inPC3 cells was greater than 80% at an mRNA concentration of 0.500 μg/mlas measured by GFP fluorescence, which is comparable to that observedwith lipofectamine administration of GFP mRNA in vitro (FIG. 17). The PKresults showed that naked mRNA was cleared rapidly with a dramaticdecrease to ˜10% after 5 min. Cy5-EGFP-mRNA-PGCP NP slightly extendedthe circulation of Cy5-EGFP-mRNA at various time points compared to thatof naked mRNA with a half-life (t_(1/2)) of >5 min, whereasCy5-EGFP-mRNA-PGDP NP had an even longer circulation profile (t_(1/2)>30min). Moreover, ˜30% of the Cy5-EGFP-mRNA PGDP NP was still circulatingafter 60 min, while naked mRNA and Cy5-mRNA-PGCP NP dropped to 1% and4%, respectively. At 240 min, 5% of the Cy5-EGFP-mRNA-PGDP NP couldstill be detected (FIG. 4A). To evaluate biodistribution (BioD) andtumor accumulation, athymic nude mice carrying human PC3 xenograft tumorwere injected with naked Cy5-EGFP-mRNA and Cy5-EGFP-mRNA NPs (both PGCPand PGDP) via tail vein. A high percentage of NPs accumulated in spleenand liver after i.v. administration. However, most importantly,Cy5-EGFP-mRNA-PGDP NP exhibited high tumor accumulation in thePC3-xenograft, whereas no or minimal signals in tumor were detected fornaked Cy5-EGFP-mRNA or Cy5-EGFP-mRNA-PGCP NP (FIG. 4B). We also examinedthe BioD of PGCP or PGDP NP encapsulating Cy5-tagged PTEN mRNA tospecifically assess the distribution of PTEN mRNA in vivo. The resultexplicitly reproduced similar BioD, exhibiting higher Cy5-PTEN-mRNAdistribution in tumor by the PGDP NP, compared to naked Cy5-PTEN-mRNA orCy5-PTEN-mRNA-PGCP NP (FIG. 18A,B). This high tumor accumulation led usto advance this platform into in vivo efficacy studies withtumor-bearing mice.

Example 6. In Vivo Therapeutic Efficacy and Mechanism of PTEN mRNA NP inPCa Xenograft Model

To validate the in vivo therapeutic efficacy of PTEN mRNA NP in PCaxenograft model, we systemically (i.v. via tail vein) injectedPTEN-mRNA-PGDP NP every three days for six injections (FIG. 5A) inimmunocompromised athymic nude mice bearing subcutaneous PC3 xenografttumors. Tumor-bearing mice injected with PBS and EGFP-mRNA-PGDP NP wereused as controls. Both PBS and EGFP-mRNA-PGDP NP groups showed rapidtumor growth, while PTEN-mRNA-PGDP NP treatment notably suppressed tumorgrowth compared to controls (FIG. 5B,C). The average tumor sizes rapidlyincreased to ˜674 mm³ and ˜738 mm³ for the controls, EGFP-mRNA-PGDP NPand PBS, respectively, which were significantly higher compared to ˜288mm³ for PTEN-mRNA-PGDP NP treatment at day 43 post tumor induction (FIG.5C). Moreover, the average tumor weight for the PTEN-mRNA-PGDP NPtreatment group was also significantly lower than that of control groups(FIG. 18C). No treatment group underwent significant changes in bodyweight, suggesting minimal toxicity (FIG. 5D). These results demonstratethe feasibility of using systemic mRNA NP delivery to reverse theeffects of tumor suppressor loss in prostate tumors in vivo.

To further understand the mechanisms underlying the therapeutic activityof PTEN-mRNA-PGDP NP, we assessed HA-PTEN expression in tumor sectionsobtained on the third day after the last injection byimmunohistochemistry analysis using HA antibody. PTEN-mRNA-PGDP NPtreatment resulted in HA-PTEN protein expression in tumor, whereas PBSand EGFP-mRNA-PGDP NP controls did not show any background PTEN-HAexpression (FIG. 5E). Next, TUNEL assay in tumor sections revealed thatPTEN-mRNA-PGDP NP increased tumor cell apoptosis significantly more thaneither EGFP-mRNA-PGDP NP or PBS. These results suggest that theeffective systemic restoration and efficient expression of PTEN intumors mediated by NP delivery of PTEN mRNA leads to enhanced tumor-cellapoptosis and decreased tumor cell survival. Consequently, this approachmay represent a viable treatment strategy for restoring tumorsuppression to PCa tumors in vivo.

Example 7. In Vivo Therapeutic Efficacy of PTEN mRNA NPs in Advanced PCaModels

To validate the in vivo therapeutic efficacy of PTEN mRNA NP in advancedPCa models, we first established a disseminated PC3 metastatic model byinjecting luciferase-tagged PC3 (PC3-luc) cells into the tail vein ofimmunocompromised, male athymic nude mice. Tumor metastases weredetected in the lung and other organs of the mice, by bioluminescenceimaging (Bruker Xtreme). Four weeks post tumor challenge, wesystemically (i.v. via tail vein) injected PTEN or EGFP mRNA-loaded PGDPNPs every three days for five injection doses and compared to PBS. Basedon the fold change of bioluminescence of PC3-luc cells in mice, it wasfound that the PTEN-mRNA-PGDP NP significantly prevented the progressionof metastatic cancer when compared to PBS and EGFP-mRNA-PGDP NPtreatment groups used as controls (FIG. 6A). Quantitative analysisdemonstrated a significant difference of the fold change in averageradiance at the experimental endpoint (day 15) in the PTEN-mRNA-PGDP NPvs. the PBS cohort (*p=0.0289), as well as the PTEN-mRNA-PGDP NP vs. theEGFP-mRNA-PGDP NP cohort (*p=0.0469) (FIG. 6B,C, FIG. 19A). Theseresults demonstrate the efficacy of the systemic delivery of PTENmRNA-loaded NPs in a disseminated metastatic PCa model.

We also tested the mRNA NPs in a PCa bone model given that bone is themost common site of PCa metastasis⁵². We performed the orthotopic,intratibial (IT) injections with the PC3-luc cells in immunocompromised,male athymic nude mice, and systemically injected PTEN-mRNA-PGDP NPs oneday post tumor inoculation and every three days for five injections intotal (FIG. 7A). Both PBS and EGFP-mRNA-PGDP NP control groupsdemonstrated rapid tumor growth (FIG. 7B,C), whereas mice receivingPTEN-mRNA-PGDP NP as compared to PBS treatment showed a significantdecrease in fold change in average radiance at the experimental endpoint(per tibia as normalized to day 0 tibia value, day 15, *p=0.0439) (FIG.7C, FIG. 19B). At the IT orthotopic model experimental endpoint (day15), cohorts which received PTEN-mRNA-PGDP NP treatment experiencedapproximately a 60% reduction in the fold change in average radiance pertibia (normalized to day 0) compared to mice receiving controlEGFP-mRNA-PGDP NP treatment (fold change for PTEN-mRNA-PGDP NPs in tibiatumor burden was 6.6, and fold change for EGFP-mRNA-PGDP NPs in tibiatumor burden was 16.3; p=0.0554). These findings indicate an ability ofPTEN-mRNA-PGDP NPs to reduce tibia tumor burden (FIG. 7C, FIG. 19B).These results are further indicative of the ability of PTEN mRNA NPdelivery to decrease tumor outgrowth in an orthotopic site of PCametastasis. Notably, no treatment group showed any significant changesof body weight in both the disseminated metastatic and IT orthotopic PCamodels, suggesting safety of this therapeutic platform (FIG. 20).

Example 8. In Vivo Safety Profile of PTEN mRNA NP

To evaluate the in vivo side effects of mRNA NPs, various organs andblood serum were harvested three days after the last injection (day 28)and at the end point (day 43) of the PCa xenograft experiment (FIG.8A,B). Organs were sectioned and H&E stained. We found no histologicaldifferences in the tissues from lung, heart, liver, spleen, or kidneybetween PBS and NP treatment groups, suggesting no notable toxicity. Forhematological analysis (days 28 and 43), we checked parameters includingaspartate aminotransferase (AST) & alanine aminotransferase (ALT) toassess liver function, creatinine and blood urea nitrogen (BUN) toevaluate kidney activity, and troponin-1 to assess cardiac functionusing appropriate assay kits. We found no obvious changes in theseparameters in serum from mice after treatment with the mRNA NPs, furtherindicating negligible side effects.

We also investigated whether mRNA NPs mediated any in vivo immuneresponse in immunocompetent mice to exclude the possibility that theanti-tumor efficacy of PTEN mRNA NP might be caused by animmunostimulatory effect. There was a similarly modest increase inlevels of proinflammatory cytokine TNF-α for both the empty PGDP NP andthe PTEN-mRNA-PGDP NP at 6 h post-injection, suggesting that thecytokine response may be attributable to the NP itself rather than theeffect of encapsulated PTEN-mRNA. PBS and naked PTEN mRNA stimulatedminimal TNF-α response as expected (FIG. 21). TNF-α levels for bothempty PGDP NPs and PTEN-mRNA-PGDP NPs returned to baseline PBS levels 24h post-injection, suggesting that immune stimulation (e.g., TNF-α) bythe NP itself was transient and that the PTEN mRNA had no lastingadverse immune/inflammatory activity. In sum, our results demonstrateeffective reversal of the PTEN-null phenotype in PCa and in breastcancer cells in vitro and in vivo after systemic delivery ofPTEN-mRNA-PGDP NPs. The inhibitory effect is dependent on the presenceof PTEN mRNA delivered by the NP and is not mediated by non-specifichost responses to either the NP itself or to the introduction of othermRNA species.

REFERENCES

-   1. Song, M. S., Salmena, L. & Pandolfi, P. P. The functions and    regulation of the PTEN tumour suppressor. Nat. Rev. Mol. Cell Biol.    13, 283-296 (2012).-   2. McCall, P., Witton, C. J., Grimsley, S., Nielsen, K. V. &    Edwards, J. Is PTEN loss associated with clinical outcome measures    in human prostate cancer? Br. J. Cancer 99, 1296-1301 (2008).-   3. Yoshimoto, M. et al. Interphase FISH analysis of PTEN in    histologic sections shows genomic deletions in 68% of primary    prostate cancer and 23% of high-grade prostatic intra-epithelial    neoplasias. Cancer Genet. Cytogenet. 169, 128-137 (2006).-   4. Han, B. et al. Fluorescence in situ hybridization study shows    association of PTEN deletion with ERG rearrangement during prostate    cancer progression. Mod Pathol 22, 1083-1093 (2009).-   5. Verhagen, P. C. et al. The PTEN gene in locally progressive    prostate cancer is preferentially inactivated by bi-allelic gene    deletion. J. Pathol. 208, 699-707 (2006).-   6. Yoshimoto, M. et al. FISH analysis of 107 prostate cancers shows    that PTEN genomic deletion is associated with poor clinical outcome.    Br. J. Cancer. 97, 678-685 (2007).-   7. Sircar, K. et al. PTEN genomic deletion is associated with p-Akt    and AR signalling in poorer outcome, hormone refractory prostate    cancer. J. Pathol. 218, 505-513 (2009).-   8. Schmitz, M. et al. Complete loss of PTEN expression as a possible    early prognostic marker for prostate cancer metastasis. Int. J.    Cancer 120, 1284-1292 (2007).-   9. Lotan, T. L. et al. PTEN protein loss by immunostaining: analytic    validation and prognostic indicator for a high risk surgical cohort    of prostate cancer patients. Clin. Cancer Res. 17, 6563-6573 (2011).-   10. Stambolic, V. et al. Negative regulation of PKB/Akt-dependent    cell survival by the tumor suppressor PTEN. Cell 95, 29-39 (1998).-   11. Furnari, F. B., Lin, H., Huang, H. S. & Cavenee, W. K. Growth    suppression of glioma cells by PTEN requires a functional    phosphatase catalytic domain. Proc. Natl. Acad. Sci. USA. 94,    12479-12484 (1997).-   12. Sun, H. et al. PTEN modulates cell cycle progression and cell    survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and    Akt/protein kinase B signaling pathway. Proc. Natl. Acad. Sci. USA.    96, 6199-6204 (1999).-   13. Suzuki, A. et al. High cancer susceptibility and embryonic    lethality associated with mutation of the PTEN tumor suppressor gene    in mice. Curr. Biol. 8, 1169-1178 (1998).-   14. Maehama, T. & Dixon, J. E. The tumor suppressor, PTEN/MMAC1,    dephosphorylates the lipid second messenger, phosphatidylinositol    3,4,5-trisphosphate. J Biol Chem 273, 13375-13378 (1998).-   15. Engelman, J. A., Luo, J. & Cantley, L. C. The evolution of    phosphatidylinositol 3-kinases as regulators of growth and    metabolism. Nat. Rev. Genet. 7, 606-619 (2006).-   16. Taylor, B. S. et al. Integrative genomic profiling of human    prostate cancer. Cancer Cell 18, 11-22 (2010).-   17. Grasso, C. S. et al. The mutational landscape of lethal    castration-resistant prostate cancer. Nature 487, 239-243 (2012).-   18. Backman, S. A. et al. Deletion of Pten in mouse brain causes    seizures, ataxia and defects in soma size resembling    Lhermitte-Duclos disease. Nat. Genet. 29, 396-403 (2001).-   19. Liliental, J. et al. Genetic deletion of the Pten tumor    suppressor gene promotes cell motility by activation of Rac1 and    Cdc42 GTPases. Curr. Biol. 10, 401-404 (2000).-   20. Tamura, M. et al. Inhibition of cell migration, spreading, and    focal adhesions by tumor suppressor PTEN. Science 280, 1614-1617    (1998).-   21. Hamada, K. et al. The PTEN/PI3K pathway governs normal vascular    development and tumor angiogenesis. Genes Dev. 19, 2054-2065 (2005).-   22. Jiang, B. H. & Liu, L. Z. PI3K/PTEN signaling in angiogenesis    and tumorigenesis. Adv. Cancer Res. 102, 19-65 (2009).-   23. Yin, H. et al. Non-viral vectors for gene-based therapy. Nat.    Rev. Genet. 15, 541-555 (2014).-   24. Quabius, E. S. & Krupp, G. Synthetic mRNAs for manipulating    cellular phenotypes: an overview. Nat. Biotechnol. 32, 229-235    (2015).-   25. Lee, J., Boczkowski, D. & Nair, S. Programming human dendritic    cells with mRNA. Methods Mol. Biol. 969, 111-125 (2013).-   26. Yamamoto, A., Kormann, M., Rosenecker, J. & Rudolph, C. Current    prospects for mRNA gene delivery. Eur. J. Pharm. Biopharm. 71,    484-489 (2009).-   27. Leonhardt, C. et al. Single-cell mRNA transfection studies:    delivery, kinetics and statistics by numbers. Nanomedicine 10,    679-688 (2014).-   28. Ligon, T. S., Leonhardt, C. & Radler, J. O. Multi-level kinetic    model of mRNA delivery via transfection of lipoplexes. PloS one 9,    e107148 (2014).-   29. Islam, M. A. et al. Biomaterials for mRNA delivery. Biomater.    Sci. 3, 1519-1533 (2015).-   30. Davis, M. E. et al. Evidence of RNAi in humans from systemically    administered siRNA via targeted nanoparticles. Nature 464, 1067-1070    (2010).-   31. Zuckerman, J. E. et al. Correlating animal and human phase Ia/Ib    clinical data with CALAA-01, a targeted, polymer-based nanoparticle    containing siRNA. Proc. Natl. Acad. Sci. USA. 111, 11449-11454    (2014).-   32. Tabernero, J. et al. First-in-humans trial of an RNA    interference therapeutic targeting VEGF and KSP in cancer patients    with liver involvement. Cancer Discov. 3, 406-417 (2013).-   33. Strumberg, D. et al. Phase I clinical development of Atu027, a    siRNA formulation targeting PKN3 in patients with advanced solid    tumors. Int. J. Clin. Pharmacol. Ther. 50, 76 (2012).-   34. Schultheis, B. et al. First-in-human phase I study of the    liposomal RNA interference therapeutic Atu027 in patients with    advanced solid tumors. J. Clin. Oncol. 32, 4141-4148 (2014).-   35. Tolcher, A. W. et al. A phase 1 study of the BCL2-targeted    deoxyribonucleic acid inhibitor (DNAi) PNT2258 in patients with    advanced solid tumors. Cancer Chemother. Pharmacol. 73, 363-371    (2014).-   36. Akinc, A. et al. A combinatorial library of lipid-like materials    for delivery of RNAi therapeutics. Nat. Biotech. 26, 561-569 (2008).-   37. Whitehead, K. A. et al. Degradable lipid nanoparticles with    predictable in vivo siRNA delivery activity. Nat. Commun. 5 (2014).-   38. Whitehead, K. A., Langer, R. & Anderson, D. G. Knocking down    barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 8,    129-138 (2009).-   39. Zuckerman, J. E. & Davis, M. E. Clinical experiences with    systemically administered siRNA-based therapeutics in cancer. Nat.    Rev. Drug Discov. 14, 843-856 (2015).-   40. Yin, H. et al. Non-viral vectors for gene-based therapy. Nature    Reviews Genetics 15, 541-555 (2014).-   41. Conde, J., Oliva, N., Atilano, M., Song, H. S. & Artzi, N.    Self-assembled RNA-triple-helix hydrogel scaffold for microRNA    modulation in the tumour microenvironment. Nat. Mater. 15, 353-363    (2016).-   42. Kauffman, K. J. et al. Optimization of Lipid Nanoparticle    Formulations for mRNA Delivery in Vivo with Fractional Factorial and    Definitive Screening Designs. Nano Lett. 15, 7300-7306 (2015).-   43. Kormann, M. S. et al. Expression of therapeutic proteins after    delivery of chemically modified mRNA in mice. Nat. Biotech. 29,    154-157 (2011).-   44. Li, B. et al. An orthogonal array optimization of lipid-like    nanoparticles for mRNA delivery in vivo. Nano Letters 15, 8099-8107    (2015).-   45. Zhu, X. et al. Long-circulating siRNA nanoparticles for    validating Prohibitin1-targeted non-small cell lung cancer    treatment. Proc. Natl. Acad. Sci. USA. 112, 7779-7784 (2015).-   46. Islam, M. A. et al. The role of osmotic polysorbitol-based    transporter in RNAi silencing via caveolae-mediated endocytosis and    COX-2 expression. Biomaterials 33, 8868-8880 (2012).-   47. Islam, M. A. et al. Accelerated gene transfer through a    polysorbitol-based transporter mechanism. Biomaterials 32, 9908-9924    (2011).-   48. Warren, L. et al. Highly efficient reprogramming to pluripotency    and directed differentiation of human cells with synthetic modified    mRNA. Cell Stem Cell 7, 618-630 (2010).-   49. Wang, Y. et al. Systemic delivery of modified mRNA encoding    herpes simplex virus 1 thymidine kinase for targeted cancer gene    therapy. Mol. Ther. 21, 358-367 (2013).-   50. Luo, X. et al. Dual-functional lipid-like nanoparticles for    delivery of mRNA and MRI contrast agents. Nanoscale 9, 1575-1579    (2017).-   51. Huang, H. et al. PTEN induces chemosensitivity in PTEN-mutated    prostate cancer cells by suppression of Bcl-2 expression. J. Biol.    Chem. 276, 38830-38836 (2001).-   52. Sturge, J., Caley, M. P. & Waxman, J. Bone metastasis in    prostate cancer: emerging therapeutic strategies. Nature reviews.    Clinical oncology 8, 357-368 (2011).-   53. Smukste, I. & Stockwell, B. R. Restoring functions of tumor    suppressors with small molecules. Cancer Cell 4, 419-420 (2003).-   54. Guo, X. E., Ngo, B., Modrek, A. S. & Lee, W. H. Targeting tumor    suppressor networks for cancer therapeutics. Curr. Drug Targets 15,    2-16 (2014).-   55. Bettinger, T., Carlisle, R. C., Read, M. L., Ogris, M. &    Seymour, L. W. Peptide-mediated RNA delivery: a novel approach for    enhanced transfection of primary and post-mitotic cells. Nucleic    Acids Res. 29, 3882-3891 (2001).-   56. Rejman, J., Tavernier, G., Bavarsad, N., Demeester, J. & De    Smedt, S. C. mRNA transfection of cervical carcinoma and mesenchymal    stem cells mediated by cationic carriers. J. Control. Release 147,    385-391 (2010).-   57. Zou, S., Scarfo, K., Nantz, M. H. & Hecker, J. G. Lipid-mediated    delivery of RNA is more efficient than delivery of DNA in    non-dividing cells. Int. J. Pharm. 389, 232-243 (2010).-   58. Read, M. L. et al. A versatile reducible polycation-based system    for efficient delivery of a broad range of nucleic acids. Nucleic    Acids Res. 33, e86 (2005).-   59. Kong, G., Braun, R. D. & Dewhirst, M. W. Hyperthermia enables    tumor-specific nanoparticle delivery: effect of particle size.    Cancer Res. 60, 4440-4445 (2000).-   60. Alexis, F., Pridgen, E., Molnar, L. K. & Farokhzad, O. C.    Factors affecting the clearance and biodistribution of polymeric    nanoparticles. Mol. Pharm. 5, 505-515 (2008).-   61. Shi, J., Kantoff, P. W., Wooster, R. & Farokhzad, O. C. Cancer    nanomedicine: progress, challenges and opportunities. Nat. Rev.    Cancer 17, 20-37 (2017).-   62. Knop, K., Hoogenboom, R., Fischer, D. & Schubert, U.S.    Poly(ethylene glycol) in drug delivery: pros and cons as well as    potential alternatives. Angew. Chem. Int. Ed. Engl. 49, 6288-6308    (2010).-   63. Guo, X. & Huang, L. Recent advances in nonviral vectors for gene    delivery. Acc. Chem. Res. 45, 971-979 (2012).-   64. Liu, H. et al. Structure-based programming of lymph-node    targeting in molecular vaccines. Nature 507, 519-522 (2014).-   65. Li, J. et al. PTEN, a putative protein tyrosine phosphatase gene    mutated in human brain, breast, and prostate cancer. Science 275,    1943-1947 (1997).-   66. Steck, P. A. et al. Identification of a candidate tumour    suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in    multiple advanced cancers. Nat. Genet. 15, 356-362 (1997).-   67. Di Cristofano, A., De Acetis, M., Koff, A., Cordon-Cardo, C. &    Pandolfi, P. P. Pten and p27KIP1 cooperate in prostate cancer tumor    suppression in the mouse. Nat. Genet. 27, 222-224 (2001).-   68. Hopkins, B. D. et al. A secreted PTEN phosphatase that enters    cells to alter signaling and survival. Science 341, 399-402 (2013).-   69. Masson, G. R., Perisic, O., Burke, I. E. & Williams, R. L. The    intrinsically disordered tails of PTEN and PTEN-L have distinct    roles in regulating substrate specificity and membrane activity.    Biochem. J. 473, 135-144 (2016).-   70. Juric, D. et al. Convergent loss of PTEN leads to clinical    resistance to a PI(3)Kalpha inhibitor. Nature 518, 240-244 (2015).-   71. Peng, W. et al. Loss of PTEN Promotes Resistance to T    Cell-Mediated Immunotherapy. Cancer Discov 6, 202-216 (2016).-   72. Campeau, E. et al. A versatile viral system for expression and    depletion of proteins in mammalian cells. PloS one 4, e6529 (2009).-   73. Ramaswamy, S. et al. Regulation of G1 progression by the PTEN    tumor suppressor protein is linked to inhibition of the    phosphatidylinositol 3-kinase/Akt pathway. Proc. Natl. Acad. Sci.    USA. 96, 2110-2115 (1999).-   74. Xu, X. et al Enhancing tumor cell response to chemotherapy    through nanoparticle-mediated codelivery of siRNA and cisplatin    prodrug. Proc. Natl. Acad. Sci. USA. 110, 18638-18643 (2013).-   75. Cox, T. R. et al. The hypoxic cancer secretome induces    pre-metastatic bone lesions through lysyl oxidase. Nature 522,    106-110 (2015).-   76. Krzywinski, M. & Altman, N. Points of significance:    Nonparametric tests. Nature methods 11, 467-468 (2014).-   77. Tammela, T. et al. A Wnt-producing niche drives proliferative    potential and progression in lung adenocarcinoma. Nature 545,    355-359 (2017).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A composition comprising one or more tumor suppressor-encoding mRNAscomplexed with a delivery vehicle.
 2. The composition of claim 1,wherein the delivery vehicle is selected from the group consisting ofprotamine complexes, lipid nanoparticles, polymeric nanoparticles,lipid-polymer hybrid nanoparticles, and inorganic nanoparticles, orcombinations thereof.
 3. The composition of claim 2, wherein thedelivery vehicle is a lipid-polymer nanoparticle.
 4. The composition ofclaim 3, wherein the core of the nanoparticle comprises a lipid, awater-insoluble polymer, and the tumor suppressor-encoding mRNAs arecomplexed with the lipid.
 5. The composition of claim 4, wherein thelipid comprises cationic lipid-like compound G0-C14.
 6. The compositionof claim 4, wherein the water-insoluble polymer comprises PLGA.
 7. Thecomposition of claim 1, wherein the tumor suppressor-encoding mRNAsencodes Phosphatase and tensin homolog on chromosome ten (PTEN).
 8. Thecomposition of claim 1, wherein the tumor suppressor-encoding mRNAsencode one or more proteins listed in Table
 1. 9. The composition ofclaim 1, wherein the tumor suppressor-encoding mRNAs comprise one ormore modifications, preferably selected from the group consisting ofARCA capping; enzymatic polyadenylation to add a tail of 100-250adenosine residues; and substitution of one or both of cytidine with5-methylcytidine and/or uridine with pseudouridine.
 10. A method oftreating a subject who has cancer, the method comprising administeringto the subject a therapeutically effective amount of the composition ofclaim
 1. 11. A method of treating a subject who has cancer, the methodcomprising administering to the subject a therapeutically effectiveamount of a composition comprising mRNA encoding Phosphatase and tensinhomolog on chromosome ten (PTEN) protein, wherein the mRNAs arecomplexed with a delivery vehicle, to a subject in need thereof.
 12. Themethod of claim 11, wherein the subject has a cancer associated withloss of PTEN expression or activity.
 13. The method of claim 12, whereinthe subject has prostate cancer, breast cancer, or glioblastoma.
 14. Themethod of claim 11, wherein the delivery vehicle is selected from thegroup consisting of protamine complexes, lipid nanoparticles, polymericnanoparticles, lipid-polymer hybrid nanoparticles, and goldnanoparticles.
 15. The method of claim 14, wherein the nanoparticle is alipid-polymer nanoparticle.
 16. The method of claim 15, wherein the coreof the nanoparticle comprises a lipid, a water-insoluble polymer, andthe tumor suppressor-encoding mRNAs are complexed with the lipid. 17.The method of claim 16, wherein the lipid comprises cationic lipid-likecompound G0-C14.
 18. The method of claim 16, wherein the water-insolublepolymer comprises PLGA.
 19. The method of claim 11, wherein the tumorsuppressor-encoding mRNAs comprise one or more modifications, preferablyselected from the group consisting of ARCA capping; enzymaticpolyadenylation to add a tail of 100-250 adenosine residues; andsubstitution of one or both of cytidine with 5-methylcytidine and/oruridine with pseudouridine.
 20. The composition of claim 1, for use intreating a subject who has cancer.
 21. The composition for the use ofclaim 20, wherein the subject has a cancer associated with loss ofexpression or activity of the tumor suppressor.
 22. The composition forthe use of claim 20, wherein the tumor suppressor-encoding mRNAscomprise mRNAs encoding Phosphatase and tensin homolog on chromosome ten(PTEN) protein, and the subject has a cancer associated with loss ofexpression or activity of PTEN.